Intracerebral hemorrhage (ICH) is a devastating stroke subtype affecting 120,000 individuals annually in the United States alone.1 Of those affected, 40% to 50% will die within the first 30 days, whereas the survivors are left with a lifetime of neurobehavioral disabilities.2 Because of the devastating impact on human health and the growing financial burden on society, an exorbitant amount of research has been put forth in an effort to determine ways in which to attenuate the devastating consequences of ICH injury. However, currently, there are no clinically viable therapeutic options for ICH patients.
One of the challenges faced with treating ICH victims has been attributed to the complexity of edema formation. After injury, close to 40% of victims experience a 1% to 2% increase in brain edema at 24 hours. This translates into a 4% to 8% increase in brain volume, leading to a severe increase in intracranial pressure and brain herniation.3 Previous studies attempting to combat the formation of edema have faced difficulties likely because of the multiple sources from which edema can develop, partly attributed to blood-brain barrier (BBB) disruption, i.e., vasogenic edema, and partly attributed to cell death, i.e., cytotoxic edema.4
Within the last decade, one of the major advances in anesthesiology has been the recognition of volatile anesthetics as neuroprotective in adults.5 Specifically, isoflurane has been acknowledged for its role in reducing apoptosis and preventing the death of key neuronal and nonneuronal cells. In a rat focal cerebral ischemic injury model, isoflurane posttreatment significantly reduced apoptosis as measured by brain infarction volume, and improved neurobehavioral deficits.6 However, to date, no study has cross-examined these potential neuroprotective effects against ICH-induced brain injury.
As a result, in this study, we investigated the potential therapeutic effects of isoflurane treatment after ICH injury. Specifically observing the potential of isoflurane to reduce apoptotic damage and ameliorate brain edema accumulation and neurobehavioral deficits, we hypothesized that 1.5% isoflurane will allow for structural preservation and, in turn, improve functional outcome after ICH injury in mice.
METHODS
This study was in accordance with the guidelines of the National Institutes of Health for the treatment of animals and was approved by the Institutional Animal Care and Use Committee at Loma Linda University. Male CD1 mice (n = 53, weight 35–45 g; Charles River, Wilmington, MA) were housed in a 12-hour light/dark cycle at a controlled temperature and humidity with free access to food and water. Mice 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).
Operative Procedure
ICH was induced using the autologous blood injection model, which was modified from previous descriptions.7–9 Briefly, mice were anesthetized with a ketamine (100 mg/kg) and xylazine (10 mg/kg) cocktail (2:1 v/v, intraperitoneal injection) and positioned prone in a stereotactic head frame (Kopf Instruments, Tujunga, CA). A scalp incision was made along the midline and a bur hole (1 mm) was drilled on the right side of the skull (0.2 mm anterior and 2.0 mm lateral of the bregma). The mouse tail was warmed with hot water for 2 minutes and then cleaned with 70% ethanol before cutting off 1.0 cm of the tail tip with sterilized surgical scissors. Next, 30 μL autologous tail blood was collected in a capillary tube without heparin and blown into a 1-mL insulin syringe. The syringe was fixed onto the microinjection pump while the needle was stereotaxically inserted into the brain through the bur hole. At first the needle was stopped at 0.7 mm above the target position and 5 μL of blood was delivered at a rate of 2 μL/min. This was done to allow for a clot to form just above the target site; that way, the chance of reflux up the needle tract is minimized. The needle was then advanced to the target position. After 7 minutes, the remaining 25 μL of blood was injected at a rate of 2 μL/min. The needle was left in place for an additional 10 minutes after injection to prevent possible leakage and withdrawn slowly over 7 minutes. After the removal of the needle, the bur hole was sealed with bone wax, the incision was closed with sutures, and the mice were allowed to recover. To avoid postsurgical dehydration, 0.5 mL normal saline was given to each mouse by subcutaneous injection after surgery.
Treatment Method
Mice were allowed to rest for 1 hour on a warm blanket before initiating therapy. Afterward, mice designated for treatment were placed into a cylindrical glass chamber containing 1.5% isoflurane carried by 30% oxygen/70% nitrogen for either 1- or 2-hour duration. The temperature was continuously monitored and maintained at 37°C. The chamber was then opened to room air where the mice were returned to their appropriate cages.
Brain Water Content Measurement
Brain water content was measured at 24 hours post-ICH injury as previously described.10–12 Briefly, mice were decapitated under deep anesthesia. Brains were immediately removed and cut into 4-mm sections. Each section was divided into 4 parts: ipsilateral and contralateral basal ganglia, and ipsilateral and contralateral cortex. The cerebellum was collected as an internal control. Tissue samples were weighed on an electronic analytical balance (APX-60; Denver Instrument, Bohemia, NY) to the nearest 0.1 mg to obtain the wet weight (WW). The tissue was then dried at 100°C for 24 hours to determine the dry weight (DW). Brain water content (%) was calculated as [(WW − DW)/WW] × 100.
Assessment of Neurobehavioral Deficits
Neurological outcomes were assessed by a blinded observer at 24 hours post-ICH using the Modified Garcia Score.13 The Modified Garcia Score is a 21-point sensorimotor assessment system consisting of 7 tests with scores of 0 to 3 for each test (maximum score = 21). These 7 tests included (1) spontaneous activity, (2) side stroking, (3) vibrissae touch, (4) limb symmetry, (5) climbing, (6) lateral turning, and (7) forelimb walking.
Immunohistochemistry
Mice (n = 12) were transcardially perfused under deep anesthesia with phosphate-buffered saline followed by 4% paraformaldehyde. The brains were then removed and postfixed in formalin. Optimum cutting temperature–embedded brains were sectioned into 10-μm-thick slices by cryostat (CM3050S; Leica Microsystems, Buffalo Grove, IL). Double immunofluorescent staining was performed using neuronal marker MAP-2 (Abcam, Cambridge, MA) and TUNEL (Roche, Indianapolis, IN). Once stained, specimens were examined under a fluorescent microscope (Olympus BX51; Melville, New York). TUNEL-positive cells were counted at the center of the hemorrhagic lesion. Three perihematomal regions of the ipsilateral cerebral hemisphere were used for cell counting. Total cell counts were converted into TUNEL-positive cell densities. Four mice per group were used for intergroup comparisons.
Statistical Analysis
All data were expressed as mean ± SEM. Statistical differences between 2 groups were analyzed using the 2-sided t test with unequal variances. Multiple comparisons were statistically analyzed with 1-way analysis of variance followed by Tukey multiple-comparison post hoc analysis or Student-Newman-Keuls test on ranks. A P value of <0.05 was considered statistically significant.
RESULTS
Isoflurane posttreatment reduces ICH-induced brain edema accumulation. Brain edema is evident in both human and experimental studies as a result of ICH.14 Accordingly, we found a significant accumulation of brain edema in the ipsilateral cortex and basal ganglia, corresponding directly to the site of injury, as compared with our nonoperated sham mice (Fig. 1). This increase in brain edema was reversed by 1-hour isoflurane posttreatment in both the ipsilateral cortex and basal ganglia (P = 0.002).
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Figure 1: Isoflurane posttreatment reduces brain edema. A, Brain edema 24 hours after operation in sham, vehicle, and isoflurane-treated mice (P = 0.002, vehicle versus treatment group). #Significant difference versus sham (P < 0.05); *significant difference versus vehicle (P < 0.05). B, Gross pictures of the mouse brain displaying the needle site with corresponding hematoma in the right basal ganglia. ICH = intracerebral hemorrhage.
Isoflurane posttreatment improves neurobehavioral deficits as assessed by the Modified Garcia Score. Surviving ICH patients are left with severe chronic neurobehavioral disabilities. The Modified Garcia Score is an accurate means of assessing both motor and sensory function.13 In our study, after ICH injury, there was a significant increase in neurobehavioral deficits compared with the sham group, with a subsequent reduction after 1-hour isoflurane posttreatment (P = 0.003; Fig. 2). Although 2-hour isoflurane posttreatment showed a trend toward improvement, it was not statistically significant (P = 0.17).
Figure 2: Isoflurane posttreatment reduces neurobehavioral deficits. A, Modified Garcia test 24 hours after operation in sham, vehicle, and isoflurane-treated mice (P = 0.003, vehicle versus 1-hour treatment group; P = 0.17, vehicle versus 2-hour treatment group). #Significant difference versus sham (P < 0.05); *significant difference versus vehicle (P < 0.05). B, Neurological outcomes were assessed by a blinded observer at 24 hours post–intracerebral hemorrhage (ICH) using the Modified Garcia Score, a 21-point sensorimotor 7-test assessment system with scores of 0 to 3 for each test (maximum score = 21).
Immunohistochemistry reveals a reduction in apoptosis after isoflurane posttreatment. In the perihematomal brain regions of the untreated mice, TUNEL was localized in circular layers of cells corresponding in size to microvascular profiles. This finding, which strongly suggests endothelial cell death, was much less abundant in the1-hour isoflurane-treated mice (Fig. 3A). To determine the neuron-specific antiapoptotic effects of isoflurane after ICH, an immunohistochemical study was undertaken using TUNEL and MAP-2 staining. Double staining revealed a marked increase in neuronal cell death after ICH injury. After 1-hour isoflurane posttreatment, TUNEL uptake in neurons was largely diminished, translating into a reduction in cell death. These results were also quantified to further support what was found qualitatively (Fig. 3B).
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 = 0.002 and 0.003 for vehicle versus treatment group). #Significant difference versus sham (P < 0.05); *significant difference versus vehicle (P < 0.05).
Reductions in cell death correlate with a reduction in neurobehavioral deficits. To better observe a correlation between cell death and neurobehavioral deficits, a separate figure plotting these 2 results against one another was created (Fig. 4). The results showed that the 2 end points, i.e., cell death and neurobehavioral deficits, are interchangeable when considering differences among groups, but seemingly independent within groups.
Figure 4: Reductions in cell death correlate with reduced neurobehavioral deficits. TUNEL+/MAP-2+ cells were plotted against neurobehavioral scores. A Lowess regression line (tension parameter of 0.5) was added to assess the shape of the relationship. ICH = intracerebral hemorrhage.
DISCUSSION
ICH is a fatal stroke subtype with no effective treatment options. Even if patients survive the initial attack, the growing hematoma triggers a series of life-threatening events leading to the accumulation of cerebral edema, progression of neurobehavioral deficits, and in some cases even death.15 In this study, we sought to determine the efficacy of isoflurane as a posttreatment therapeutic modality for ICH brain injury. We found that 1-hour 1.5% isoflurane posttreatment resulted in a significant reduction in brain edema, a qualitative decrease in apoptotic cell death (including both neuronal and nonneuronal cells), and a significant improvement in neurobehavioral deficits. To the best of our knowledge, this is the first study demonstrating the effectiveness of isoflurane posttreatment after ICH injury by reducing structural damage and subsequently preserving functional integrity.
The movement of water from the vasculature to the brain is highly regulated by the BBB, which maintains both a physiologic and physical barrier, thereby preventing the accumulation of fluid in the injured brain. During an ICH injury, the BBB is disrupted by a variety of products including inflammatory mediators (i.e., infiltrating immune cells), thrombin, hemoglobin breakdown products, oxidative stress, and matrix metalloproteinases signals.14 A disruption in BBB patency leads to dysregulation of water homeostasis between the vasculature and brain parenchyma. This translates into severe increases in intracranial pressure, a reduction in cerebral blood flow, subsequent increase in ischemic damage, and further increase in cerebral edema accumulation secondary to apoptotic cell death.16 Clinically, this means an increased risk for herniation from the ensuing increase in intracranial pressure as well as severe neurobehavioral deficits from neuronal and nonneuronal cell death.
Similar to what we would expect physiologically in the ICH patient, we found a significant increase in brain edema accumulation between our nonoperated sham group and ICH-injured group. Two sources likely contributed to the accumulation of edema in our ICH population: first, from the disruption of the BBB facilitating increases in vascular permeability from endothelial cell death; and second, from direct neuronal and nonneuronal cell deaths observed on TUNEL and MAP-2 staining. These effects were subsequently ameliorated after 1-hour 1.5% isoflurane posttreatment, suggesting that isoflurane has a key role in reducing apoptosis after cerebrovascular injury. Additionally, the preservation of structural integrity translated into a significant improvement in neurobehavioral deficits. These results are in line with previous work that has also suggested an antiapoptotic role for isoflurane.17
Previous studies have implied that through modulation of intracellular calcium levels, isoflurane has been implicated in indirect activation of antiapoptotic factors including Ca/calmodulin-dependent protein kinase II, the phosphatidylinositol-3-kinase (PI3K)/AKT cascade, and mitogen-activated protein kinase.18,19 However, it is our belief that isoflurane works to block the release of proapoptotic factors intracellularly through another mechanism. Activation of the PI3K signaling pathway promotes cell survival. One way it does so is through phosphorylation inhibition of glycogen synthase kinase 3β (GSK3β), a key serine/threonine protein kinase that regulates the opening of the mitochondrial permeability transition pores. We hypothesize that by inhibiting the activation of GSK3β indirectly by isoflurane's activation of PI3K, the mitochondrial permeability transition pore is unable to release proapoptotic factors that normally occur with neuronal cell environment disruption. Although we understand this is simple speculation until further mechanistic studies are conducted and furthermore a limitation in our study, we do acknowledge the work that has been previously published on myocardial ischemia models and isoflurane that supports this mechanistic theory.20
An additional question that would need to be addressed through further studies would be the optimal duration time for posttreatment with isoflurane. In our study, we found that only 1-hour isoflurane posttreatment significantly improved our measured outcomes whereas 2-hour treatment failed to do so. This could be attributed to isoflurane's ability to increase cerebral blood flow, whereby 1 hour of therapy is just enough to balance an increase in blood flow with a reduction in penumbral ischemic damage, whereas longer durations can in fact enhance or exacerbate damage. Because this is only speculative, further studies will be needed to determine exactly how isoflurane provides neuroprotection and determine optimal therapy durations. An additional question that would need to be addressed in further studies would be the effects of isoflurane posttreatment on long-term outcomes. Currently, our study only evaluated the short-term benefits of isoflurane; future investigations would need to determine whether the neuroprotection afforded by isoflurane at 24 hours can be sustained at longer time points.
Isoflurane has come under intense scrutiny from studies suggesting that it only protects the brain acutely and postpones or delays the onset of injury.21 However, the evidence at large remains contradictory and a host of studies both affirm and refute the notion of injury postponement.6,21 As demonstrated in this study, low-dose isoflurane posttreatment may be effective at protecting the injured hemorrhagic brain and may be a promising therapeutic option as an acute treatment after ICH injury.
DISCLOSURES
Name: Nikan H. Khatibi, MD, MBA.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Nikan H. Khatibi has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Qingyi Ma, BS.
Contribution: This author helped conduct the study.
Attestation: Qingyi Ma has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: William Rolland, BS.
Contribution: This author helped conduct the study.
Attestation: William Rolland has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Robert Ostrowski, MD, PhD.
Contribution: This author helped conduct the study.
Attestation: Robert Ostrowski has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Nancy Fathali, PhD.
Contribution: This author helped conduct the study and write the manuscript.
Attestation: Nancy Fathali has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Robert Martin, MD.
Contribution: This author helped design the study and write the manuscript.
Attestation: Robert Martin has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Richard Applegate, MD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Richard Applegate has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Gary Stier, MD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Gary Stier has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Jiping Tang, MD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Jiping Tang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: John H. Zhang, MD, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: John H. Zhang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
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