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Effect of Delayed Mild Brain Hypothermia on Edema Formation After Intracerebral Hemorrhage in Rats
Effect of Delayed Mild Brain Hypothermia on Edema Formation After Intracerebral Hemorrhage in Rats Masahiko Kawanishi, MD,* Nobuyuki Kawai, MD,* Takehiro Nakamura, MD,† Chengyi Luo, MD,* Takashi Tamiya, MD,* and Seigo Nagao, MD*
Secondary consequences of intracerebral hemorrhage (ICH) including inflammation, edema, and oxidative damage all contribute to cell death after ICH. Brain hypothermia (BH) has been used as an effective neuroprotective treatment in experimental brain ischemia and traumatic brain injury. In this study, we first attempted to evaluate the effect of delayed mild BH (35 C) on brain edema formation 48 hours after ICH. BH was started 3, 6, 12, and 24 hours after the induction of 100 mL of autologous blood into the basal ganglia (hypothermic [HT]; HT3: n 5 4, HT6: n 5 6, HT12: n 5 11, HT24: n 5 6) in rats. To examine the protective mechanism of BH, blood-brain barrier (BBB) permeability to Evans blue, accumulation of polymorphonuclear leukocyte, and oxidative DNA damage in the lesion were compared between normothermic (NT) (37 C) and HT6 rats 48 hours after ICH. Finally, neurologic recovery was assessed using behavioral tests in NT and HT6 rats 48 hours after ICH. Brain water content in the ispilateral basal ganglia was significantly reduced with delayed BT compared with NT (n 5 7, 81.8 6 0.7% v HT3: 78.9 6 0.8%, P , .01; HT6: 78.7 6 0.6%, P , .01; HT12: 79.4 6 1.1%, P , .01; HT24: 80.3 6 0.6%, P , .01). The BBB disruption to Evans blue was significantly reduced with BH (HT6: n 5 6) compared with NT (n 5 6) rats in the ipsilateral basal ganglia (23.0 6 5.2 v 42.3 6 4.0 ng/g wet tissue, P , .05). HT6 treatment (n 5 6) significantly inhibited the accumulation of polymorphonuclear leukocyte compared with NT treatment (n 5 6) (0.43 6 0.22 v 1.49 6 0.61 DAbs/mg tissue, P ,.05). HT6 treatment (n 5 3) also significantly reduced oxidative DNA damage determined with 8-hydroxyl-2’-deoxyguanosine compared with NT treatment (n 5 3) (92 6 18 v 40 6 7 pg 8-hydroxyl-2’-deoxyguanosine/mg DNA, P , .05). Furthermore, HT6 treatment (n 5 5) significantly improved neurologic recovery assessed with forelimb placing score compared with NT treatment (42.0 6 5.8 v 12.0 6 3.7, P , .05). In conclusion, mild BH significantly reduces the brain edema formation after ICH, even when the BH is applied 24 hours after hematoma induction in rats. Several neuroprotective mechanisms, including reduced BBB disruption, inflammation and oxidative damage, are suggested in this study. Key Words: Blood-brain barrier—brain edema—brain hypothermia—intracerebral hemorrhage—oxidative damage—polymorphonuclear leukocyte. Ó 2008 by National Stroke Association
From the Departments of *Neurological Surgery and †Neurobiology, Kagawa University School of Medicine, Kagawa, Japan. Received October 12, 2007; revision received January 7, 2008; accepted January 30, 2008. Address correspondence to Nobuyuki Kawai, MD, Department of Neurological Surgery, Kagawa University School of Medicine, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. E-mail: nobu@med. kagawa-u.ac.jp. 1052-3057/$—see front matter Ó 2008 by National Stroke Association doi:10.1016/j.jstrokecerebrovasdis.2008.01.003
Spontaneous intracerebral hemorrhage (ICH) causes high mortality and poor neurologic recovery even in survivors. In ICH, significant tissue damage occurs immediately because of space-occupying effects and secondary consequences including brain edema formation contribute further tissue damage to cell death. Patients with ICH deteriorate progressively as a result of ongoing brain edema. A number of mechanisms may be involved in the brain edema formation after ICH.1 Brain edema after ICH
Journal of Stroke and Cerebrovascular Diseases, Vol. 17, No. 4 (July-August), 2008: pp 187-195
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was believed to be caused by local pressure compressing the microcirculation and producing ischemia in the region around the hematoma.2 However, there is no experimental evidence that the degree or duration of hypoperfusion around the hematoma is sufficient to produce ischemic brain damage.3 Attention later focused on the direct role of the biochemical substances from an intracerebral blood clot in generating brain edema. At least two phases of edema formation are involved in ICH.1 These include an acute phase (first 2 days) involving the activation of the coagulation cascade and thrombin formation and a subacute phase (after 3 days) involving red blood cell lysis and hemoglobin-induced neuronal toxicity.4-6 Iron is the hemoglobin degradation product and iron overload in the brain can cause free radical formation and oxidative damage such as lipid peroxidation after ICH.7-9 Inflammatory reactions including leukocyte accumulation and activation of the complimentary system in the brain parenchyma also play an important role in ICH-related brain edema formation.10,11 Mild, prolonged brain hypothermia (BH) improves outcome in rodent models of global and focal ischemia. Clinically, BH can be safely applied to patients with severe head trauma and stroke. Recently, renewed interest has been directed to global ischemia in resuscitated patients with cardiac arrest and clinical trials report improved outcome when prolonged BH is induced soon after cardiac arrest.12,13 BH may reduce secondary consequences of ICH such as edema, blood-brain barrier (BBB) disruption, inflammation, and oxidative damage in the perihematomal region. Early work from our laboratory shows that immediate BH reduces vasogenic edema formation after intrastriatal thrombin injection in rats.14 Recently, MacLellan et al15 revealed that BH initiated 1 or 4 hours after autologous blood infusion into the striatum reduces brain edema formation, BBB disruption, and inflammatory cell infiltration. We hypothesized that delayed BH would improve outcome in the whole blood model of ICH because progression of brain injury around hematoma is an ongoing phenomenon.1 In this study, we assessed whether delayed post-ICH BH affects the brain edema formation, BBB permeability, inflammation, oxidative damage, and behavioral recovery.
Materials and Methods The experimental protocols used in this study were approved by our ethics committees for animal experiment. Animals were allowed free access to food and water before the experiment.
(60 mg/kg). A polyethylene (PE-50) catheter was inserted into the femoral artery to monitor the arterial blood pressure and to obtain arterial blood for analysis of blood gases, blood pH, hematocrit, and blood glucose. The rat’s body (rectal) temperature was maintained at 37 C during the surgery using a feedback-controlled heating system. The rats were positioned in a stereotactic frame and the scalp was incised along the midline. Using a sterile technique, a 1 mm burr hole was opened in the skull on the left coronal suture 3 mm lateral to the midline. A blunt 26-gauge needle was inserted into the left basal ganglia under stereotactic guidance (coordinates: 0.2 mm anterior, 6.0 mm ventral, and 3.0 mm lateral to the bregma). Then, a 100 mL of autologous whole blood was infused at a rate of 20 mL/min with the use of a microinfusion pump (EPS-26, Eicom, Kyoto, Japan). After completion of the infusion, the needle was withdrawn quickly and cyanoacrylate glue was placed around the burr hole, and the skin incision was closed with sutures. After the surgery, rats were divided into normothermic (NT) and hypothermic (HT) groups. NT rats were housed in a room maintained at 25 C for 48 hours and HT rats were housed in a cold room maintained at 5 C with fine water misters for cooling. HT treatment was started 3 (HT3), 6 (HT6), 12 (HT12), and 24 (HT24) hours after the induction of ICH. In a pilot study, we examined what degree of brain temperature was produced in a room maintained at 25 C (n 5 5) and in a cold room maintained at 5 C (n 5 5) in awake rats using a telemetry system. A needle-type thermotelemeter (TA10TA-F20, Data Sciences, St. Paul, Minn) was inserted into the brain at a 6-mm depth on the coronal suture. The data from the thermotelemeter were sent to the telemetry receiver (RPC-1, Data Sciences) and continuously monitored for 48 hours.
Experimental Protocols This study was divided into 3 parts. The first part evaluated the effect of brain temperature on the brain edema formation 48 hours after ICH in NT (n 5 7) and HT (HT3: n 5 4, HT6: n 5 6, HT12: n 5 11, HT24: n 5 6) rats. In the second part, the BBB permeability to Evans blue (EB) dye, accumulation of the polymorphonuclear (PMN) leukocyte, and oxidative DNA damage in the lesion 48 hours after ICH were examined in NT and HT6 rats to clarify the protective mechanism of BH. In this part, 6 rats were used for EB and PMN studies and 3 rats were used to study oxidative damage in each group. Finally, neurologic recovery was assessed using two behavioral tests in NT (n 5 5) and HT6 (n 5 5) rats 24 and 48 hours after ICH.
Animal Preparation and Intracerebral Blood Infusion Adult male Sprague-Dawley rats (Charles River Laboratories, Hino, Japan) weighing 250 to 400 g were anesthetized with intraperitoneal sodium pentobarbital
Measurement of Brain Water Content The rats were killed by decapitation under deep pentobarbital anesthesia 48 hours after ICH induction. The
DELAYED BRAIN HYPOTHERMIA FOR INTRACEREBRAL HEMORRHAGE
brains were removed rapidly and two coronal slices 3 mm from the frontal pole were cut 3-mm thick. The anterior slice (slice A) contained the needle trajectory and arterial blood injection site at the basal ganglia. The adjacent slice (slice B) was a section posterior to the arterial blood injection site. These sections of brain were divided along the midline and the cortex was separated from the basal ganglia bilaterally. The hematoma itself in the basal ganglia was removed gently from the surrounding tissue. The tissue samples were immediately weighed on an electronic analytic balance to the nearest 0.1 mg to obtain the wet weight (WW). The tissue was then dried in an oven at 110 C for 24 hours and weighed again to obtain the dry weight. The formula, (WW – dry weight)/WW 3 100, was used to calculated the water content and expressed as percentage WW.
BBB Permeability to EB BBB integrity was investigated using EB dye extravasation. EB solution (Sigma Chemical Co, St Louis, Mo) (2% in saline; 2 mL/kg) was injected intravenously 48 hours after ICH induction and allowed to circulate for 30 minutes under sodium pentobarbital anesthesia (60 mg/kg). The chest was subsequently opened and the brain was transcardially perfused with 200 mL of saline through the left ventricle at 100-mm Hg pressure until almost colorless perfusion fluid was obtained from the right atrium. After decapitation, the brains were removed and two coronal slices (slices A and B) 3 mm from the anterior pole were cut 3-mm thick. These sections of the brain were divided along the midline and the cortex was separated from the basal ganglia. The hematoma in the basal ganglia was removed from the surrounding tissue. Samples were then weighed and placed in 50% trichloroacetic solution. After homogenization and centrifugation, the extracted dye was diluted with ethanol (1:3), and its fluorescence was determined (excitation at 620 nm and emission at 680 nm) with a luminescence spectrophotometer (65040, Hitachi, Tokyo, Japan). Calculations were based on an external standard (62.5-500 ng/mL) in the same solvent. The tissue content of EB was quantified from a linear standard line and was expressed per gram of tissue.
Measurement of Myeloperoxidase Activity Myeloperoxidase (MPO) activity was used as an indicator of tissue PMN leukocyte accumulation. The brains were transcardially perfused with 200 mL of cold saline through the left ventricle at 100-mm Hg pressure and removed rapidly. A coronal slice 3 mm from the frontal pole was cut 5-mm thick and separated into right and left hemispheres. The hematoma was removed from the basal ganglia. Each hemisphere was immediately frozen in liquid nitrogen and stored at –80 C for later analysis. The procedure described by Bradley et al16 was used for the quantification of MPO activity with minor modification.
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The brain samples were thawed on ice and the cortex was separated from the basal ganglia. The WW of each sample was immediately measured. Each sample was homogenized in 5-mmol/L potassium phosphate buffer (pH 6.0, 4 C) at an original tissue weight:volume ratio of 1:20 and centrifuged at 30,000 3 g (30 minutes, 4 C). The supernatant was discarded and the pellet was washed again as described above. After decanting the second supernatant, the pellet was extracted by suspension in 0.5 hexadecyltrimethylammonium bromide in 50 mmol/L potassium phosphate buffer (pH 6.0, 25 C) for approximately 2 minutes at an original tissue weight:volume ratio of 1:10. The samples were immediately frozen in liquid nitrogen. Three freeze/thaw cycles were performed with sonications (10 seconds, 25 C) between cycles. After the last sonication, the samples were incubated at 4 C for 20 minutes and centrifuged at 12,500 3 g (15 minutes, 4 C). A 0.1-mL aliquot of supernatant was mixed with 2.9 mL of 50-mmol/L potassium phosphate buffer, pH 6.0, containing 0.167 mg/mL of o-dianisidine dihydrochloride and 0.005% hydrogen peroxide. The change in absorbance at 460 nm during 2 minutes was measured by a spectrophotometer (U-3300, Hitachi). All chemicals used in this study were supplied by Sigma Chemical Co.
Measurement of 8-Hydroxyl-2’-Deoxyguanosine in DNA DNA is vulnerable to oxidative stress. Deoxyguanosine residues in DNA are hydroxylated by various agents that produce free radicals resulting in 8-hydroxyl-2’-deoxyguanosine (8-OHdG) accumulations. 8-OHdG might serve as a sensitive biomarker of intracellular oxidative stress.17 Brains were treated rapidly and stored as described in the MPO study. DNA extraction was performed using a DNA isolation kit (Dojindo Molecular Technologies, Kumamoto, Japan). The levels of 8-OHdG of the samples were determined using enzyme-linked immunosorbent assay kit (Japan Institute for the Control of Aging, Shizuoka, Japan). The kit measures extremely low levels of 8-OHdG, and the specificity of the monoclonal antibody has been established. The wells were subjected to optical density measurement at 450 nm. 8-OHdG enzyme-linked immunosorbent assay was triplicate and the means were calculated. The data, expressed as pg 8-OHdG/mg DNA, were calculated based on the linear calibration curve generated for each experiment using 8-OHdG standard solutions.
Behavioral Tests (Forelimb Placing Test and Corner Turn Test) Forelimb placing was scored using the vibrissae-elicited forelimb placing test. Animals were held by their bodies to allow their forelimbs to hang free. Independent testing of each forelimb was induced by brushing the
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Table 1. Physiologic parameters
No. of animals MABP (mm Hg) pH PaCO2 (mm Hg) PaO2 (mm Hg) Hematocrit (%) Blood glucose (mg/dL)
NT
HT3
HT6
HT12
HT24
7 114 6 8 7.42 6 0.05 42 6 8 96 6 11 42 6 4 106 6 19
6 108 6 10 7.43 6 0.06 44 6 6 85 6 9 44 6 5 99 6 16
6 111 6 6 7.44 6 0.04 43 6 4 89 6 7 44 6 6 103 6 21
11 102 6 7 7.47 6 0.06 41 6 5 94 6 14 43 6 5 103 6 15
6 103 6 8 7.41 6 0.05 45 6 5 84 6 8 44 6 4 97 6 20
Abbreviations: HT, hypothermia treatment started 3 (HT3), 6 (HT6), 12 (HT12), and 24 (HT24) hours after induction of intracerebral hemorrhage; MABP, mean arterial blood pressure; NT, normothermia. Measurements were performed in each group at the time of autologous blood injection. Values are expressed as mean 6 SD.
Statistical Analysis Differences in the brain edema formation among groups (NT, HT3, HT6, HT12, and HT24) were analyzed using analysis of variance and the Scheffe´ F test of significance. The physiologic variables, EB extravasations, MPO activities, 8-OHdG levels, and behavioral scores (corner turn score and forelimb placing score) in the NT and HT6 groups were compared by unpaired Student’s t test. A two-tailed probability value of less than .05 was used to indicate a significant difference. All values are expressed as mean 6 SEM.
Results Physiologic Parameters Table 1 shows the mean values of physiologic parameters in NT and HT (HT3, HT6, HT12, and HT24) rats used in the brain water content study. There were no significant differences in the values of arterial blood pressure, blood gas tensions, hematocrit level, and blood glucose concentration at the time of autologous blood injection among the groups. According to the pilot study, the brain temperature in rats housed in a cold room maintained at 5 C reduced
temporarily to about 30 C and then gradually recovered to 35 C, which lasted throughout the observation (Fig 1).
Brain Water Content Intracerebral injection of a 100-mL autologous blood produced medium ICH in the basal ganglia, which sometimes extended to the lateral ventricle (Fig 2, A). BH treatment significantly reduced the brain edema formation in the ipsilateral basal ganglia (NT: 81.8 6 0.7% v HT3: 78.9 6 0.8%, P , .01; HT6: 78.7 6 0.6%, P , .01; HT12: 79.4 6 1.1%, P , .01; HT24: 80.3 6 0.6%, P , .01) (Fig 3, A) and cortex (NT: 81.0 6 1.0% v HT3: 79.6 6 0.9%, P , .05; HT6: 79.1 6 0.9%, P , .01; HT12: 79.3 6 0.8%, P , .01; HT24: 79.5 6 0.3%, P , .01) (Fig 3, B) in the slice of blood injection (slice A). An increase in brain water content was also significantly suppressed with BH in the ipsilateral basal ganglia (NT: 80.9 6 0.9% v HT3: 78.5 6 0.3%, P , .01; HT6: 78.9 6 0.4%, P , .01; HT12: 79.5 6 0.9%, P , .01; HT24: 79.7 6 0.8%, P , .05) (Fig 3, C) and cortex (NT: 79.5 6 0.9% v HT3: 78.6 6 0.6%, P , .05; HT6: 78.6 6 0.4%, P , .05; HT12: 78.8 6 0.8%, P , .05; HT24: 78.8 6 0.6%, P , .05) (Fig 3, D) in the slice adjacent 45
Brain temperature (°C)
respective vibrissae on the corner of a table top once per trial for 10 trials. A score of one was given each time the rat placed its forelimb onto the edge of the table in response to the vibrissae stimulation. Percentage success in placing responses was determined for impaired forelimb and nonimpaired forelimb. Corner turn test was also used in this study. For the corner turn test, the rat was allowed to proceed into a corner, the angle of which was 30 degrees. To exit the corner, the animal could turn either to the left or right, and this was recorded. This was repeated 10 to 15 times, and the percentage of right turns was calculated. Both forelimb placing and corner turn tests were performed and scored by an investigator blind to the treatment conditions.
Normo Hypo 40
35
30
25
0
4
8
12
16
20
24
28
32
36
40
44
48
Time course (hours) Figure 1. Line graphs depicting time-course changes in brain temperature in rats housed in room maintained at 25 C (normo) (n 5 5) and in cold room maintained at 5 C (hypo) (n 5 5). Measurements were made in awake rats using telemetry system for 24 hours.
DELAYED BRAIN HYPOTHERMIA FOR INTRACEREBRAL HEMORRHAGE
191
Figure 2. (A) Photographs showing coronal sections (2-mm thick) containing hematoma 24 hours after autologous blood infusion. (B) Photographs showing Evans blue staining observed in basal ganglia adjacent to hematoma (small arrows) and overlying ipsilateral cortex (large arrows).
to blood injection (slice B). Differences in the brain water contents were not statistically significant among the HT subgroups.
BBB Permeability to EB As shown in Fig 4, EB staining was observed in the basal ganglia adjacent to the hematoma and overlying ipsilateral cortex, indicating increased BBB permeability and vasogenic edema development (Fig 2, B). The BBB was significantly protected from breakdown with BH compared with NT rat in the ispilateral basal ganglia (NT: 42.3 6 4.0 v HT6: 23.0 6 5.2 ng/g wet tissue, P , .05), but not in the ipsilateral cortex (NT: 53.3 6 10.6 v HT6: 33.8 6 10.4 ng/g wet tissue) in the slice of blood injection (slice A). In the adjacent slice B, the BBB disruption was significantly inhibited with BH compared with NT in the ipsilateral basal ganglia (NT: 55.5 6 9.3 v HT6: 21.8 6 3.2 ng/g wet tissue, P , .05) and cortex (NT: 34.9 6 9.3 v HT6: 16.6 6 3.5 ng/g wet tissue, P , .01).
MPO Activity As shown in Fig 5, A, using light microscopy, a prominent perihematomal accumulation of PMN leukocytes was observed in NT rats 48 hours after ICH induction. The MPO activity is used as an indicator of tissue PMN leukocyte accumulation. There was a significant effect with BH on tissue MPO activity. BT significantly reduced the accumulation of PMN leukocytes in the ipsilateral hemisphere compared with NT rats (NT: 1.49 6 0.61 v HT6: 0.43 6 0.22 DAbs/mg tissue, P , .05). A significant difference was not observed in the contralateral hemisphere between the NT and HT6 groups.
8-OHdG in DNA As shown in Fig 5, B, 8-OHdG is formed from deoxyguanosine in DNA by hydroxyl free radicals and might serve as a sensitive biomarker of intracellular oxidative stress. 8-OHdG level in the extracted DNA is significantly lower in the ipsilateral hemisphere treated with BH
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A85
B 85 NT HT3
83
HT6
81 79
**
**
HT12 HT24
** **
77 75
HT12
** ** **
HT24
79 77
Ipsilateral
Contralateral
D 85 HT6
81
** ** **
*
Cortex: slice B
NT HT3
83
HT12 HT24
77 75
Brain Water Content (%)
BG: slice B
Brain Water Content (%)
HT6
*
81
Contralateral
85
79
HT3
83
75
Ipsilateral
C
NT
Cortex: slice A
Brain Water Content (%)
Brain Water Content (%)
BG: slice A
NT HT3
83
HT6
81
HT12
*
79
*
HT24
* *
Figure 3. Bar graph comparing brain water content between NT (n 5 7) and HT (HT3: n 5 4, HT6: n 5 6, HT12: n 5 11, HT24: n 5 6) rats in slice of hematoma injection ([A] basal ganglia [BG], [B] cortex) and in adjacent slice ([C] BG, [D] cortex) 48 hours after ICH. Hypothermia significantly reduced brain water content in ipsilateral BG and cortex in slice of hematoma injection compared with NT rats. In adjacent slice, HT also significantly reduced brain water content in ipsilateral BG and cortex in slice of hematoma injection. Differences in brain water content were not significant among HT subgroups (HT3, HT6, HT12, and HT24). **P , .01 and *P , .05.
77 75
Ipsilateral
Contralateral
Ipsilateral
compared with that in NT rats (NT: 92 6 18 v HT6: 40 6 7 pg/mg DNA, P , .05). A significant difference was not observed in the contralateral hemisphere between the NT and HT6 groups.
Contralateral
when the hematoma mass initially compressed the brain resulting in microcirculatory disorder with consequent ischemia in the brain tissue surrounding the hematoma.2 However, there is no experimental evidence that the degree or duration of hypoperfusion around the hematoma
Behavioral Tests
Discussion ICH is a common emergency stroke treated by neurologists and neurosurgeons, and its neurologic manifestations may frequently exacerbate as a result of brain edema after initial brain damage with hematoma. Brain edema after ICH gradually develops within 24 hours after the ictus and continues to develop for the next couple of days.18 The underlying mechanism of brain edema formation after ICH is complex and has not been fully elucidated.1 Brain edema after ICH was believed to occur
70 NT
BBB Permeability to Evan’s Blue (ng / g wet tissue)
As shown in Fig 6, neurologic deficits were examined using the forelimb placing test and corner turn test 24 and 48 hours after ICH. The forelimb placing score was significantly improved with BH initiated 6 hours after ICH compared with NT rats 48 hours after ICH (NT: 12 6 8 v HT6: 42 6 13%, P , .05), but not 24 hours after ICH (NT: 2 6 4 v HT6: 14 6 13%, P 5 .09). On the other hand, the corner turn score was not significantly different between the NT and HT6 groups examined at 24 and 48 hours after ICH induction.
60
HT6
50 40
*
30
* **
20 10 0 Cortex
BG
Slice A
BG
Cortex
Slice B
Figure 4. Bar graph comparing BBB permeability to EB between NT (n 5 6) and HT (HT6: n 5 6) rats in slice of hematoma injection (slice A) and in adjacent slice (slice B) 48 hours after ICH. Hypothermia significantly reduced BBB permeability to EB in ipsilateral basal ganglia (BG) in slice A and in ipsilateral BG and cortex in slice B compared with NT group. **P , .01 and *P , .05.
DELAYED BRAIN HYPOTHERMIA FOR INTRACEREBRAL HEMORRHAGE
A
193
B
2.5
120
2
1.5
1
* 0.5
125 NT
Forelimb Placing Score (%)
60
* 40
0
is sufficient to produce ischemic brain damage.3 Several studies have demonstrated that toxic substances released from the clot are involved in accelerating the brain injury.4-6 These include the acute phase (first 2 days) involving the activation of the coagulation cascade and thrombin formation and the subacute phase (after 3 days) involving red blood cell lysis and hemoglobininduced neuronal toxicity. Thrombin is involved in the development of brain injury after ICH.4 Thrombin induces BBB disruption and the death of parenchymal cells directly.19 There is also evidence that thrombin has effects related to inflammation with chemotaxis of leukocytes, activation of microglia, and cytokine release.14,20,21 Red blood cell lysis and hemoglobin toxicity also lead to
HT6
75
* 50
25
0 24 hours
80
20
ipsilateral
Before
HT6
HT6
0
100
NT 100
8-OHdG (pg / µg DNA)
MPO Activity (
Figure 5. (A) Bar graph comparing MPO activity in ispilateral hemisphere between NT (n 5 6) and HT (HT6: n 5 6) rats 48 hours after ICH. HT treatment significantly reduced PMN leukocyte accumulation compared with NT. (B) Bar graph comparing 8-OHdG level in DNA extraction between NT (n 5 3) and HT (HT6: n 5 3) rats 48 hours after ICH. HT treatment significantly reduced 8-OHdG concentration (pg) per mg DNA in ipsilateral hemisphere compared with NT. (A and B) Significant difference was not observed in contralateral hemisphere between two groups. *P , .05.
Abs / mg tissue)
NT
48 hours
Figure 6. Bar graph comparing forelimb placing score between NT (n 5 5) and HT (HT6: n 5 5) rats 24 and 48 hours after ICH. Forelimb placing score was significantly improved with HT treatment compared with NT 48 hours after ICH, but not 24 hours after ICH. *P, .05.
contralateral
ipsilateral
contralateral
secondary injury after ICH.1,5,6 Iron is a hemoglobin degradation product and iron overload can lead to detrimental results on the brain tissue.7-9 A previous study showed that intracerebral infusion of iron causes brain edema, and that an iron chelator, deferoxamine, reduces hemoglobin-induced brain edema, indicating that iron plays an important role in edema formation after ICH.22 A recent study demonstrates that infusion of autologous whole blood and ferrous iron into the basal ganglia causes oxidative DNA damage, suggesting that iron might contribute to ICH-induced oxidative stress.23 Mild, prolonged BH improves functional recovery and reduces injury after global and focal ischemia in rodents. Mild BH is a safe and feasible treatment for head injury and stroke victims. Notably, recent clinical trials report improved outcome when prolonged BH is induced in resuscitated patients with cardiac arrest.12,13 A number of underlying mechanisms have been proposed in the neuroprotective effects of BH in experimental ischemic brain injury. Secondary consequences of ischemia and ICH include inflammation, edema, and oxidative stress, which all lead to cell death. Given the overlap in mechanisms contributing to injury after ischemia and ICH, it makes sense to apply BH to ICH. Our previous study revealed that early BH reduces the BBB disruption, PMN leukocyte accumulation, and brain edema after intrastriatal thrombin injection in rats.14 Unexpectedly, however, MacLellan et al15 demonstrated that BH initiated soon after collagenase-induced ICH fails to reduce tissue damage or behavioral deficits. This result is attributable to general side effects of BH (e.g., elevated blood pressure, coagulopathy), which would counteract beneficial effects of BH during the early post-ICH period. Later, they showed that BH delayed 12 hours after collagenase-induced ICH improves recovery and lessens tissue loss because
M. KAWANISHI ET AL.
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bleeding would not be aggravated at this time. Recently, MacLellan et al15 revealed that BH initiated 1 or 4 hours after autologous blood infusion into the striatum reduces brain edema formation, BBB disruption, and inflammatory cell infiltration. We hypothesized that delayed BH would improve outcome in the whole blood ICH model because progression of brain injuries around hematoma is an ongoing phenomenon. In this study, we clearly demonstrate that delayed BH initiated 3 to 24 hours after ICH induction exhibits protective effect in rats. Our study also revealed that delayed BH initiated 6 hours after ICH reduced PMN leukocyte accumulation and oxidative DNA damage in the lesion. These are not a result of general physiologic effects of BH on the brain because a significant difference was not observed in the contralateral hemisphere between NT and HT rats, indicating that HT per se does not influence the PMN infiltration and oxidative DNA damage in the normal brain tissue. Previous studies have shown that the treatment time window for BH in experimental ischemic and traumatic brain injuries is narrow.25,26 In this model of ICH, an early study demonstrated that the brain edema in the basal ganglia develops progressively over 24 hours and remains relatively constant for 4 days before beginning to resolve.3 It is reported that BH can promptly reduce BBB breakdown after traumatic brain injury in rats.27 Therefore, BH applied 24 hours after ICH induction could improve perihematomal BBB damage and reduce vasogenic brain edema in the basal ganglia. There are some limitations in this study. First, we did not determine whether delayed BH provides long-lasting neuroprotection in rats with ICH. BH may have postponed harmful processes around hematoma as found in cerebral ischemia, in which BH sometimes delays inflammation and neuronal damage.28 A previous study demonstrated that BH started 12 hours after collagenase injection into the basal ganglia reduces chronic tissue loss 30 days after ICH.24 However, a recent study shows that BH started 1 and 4 hours after autologous whole blood infusion into the striatum does not affect lesion volume 30 days after ICH induction.15 In the whole blood infusion ICH model, the initial volume of tissue damage in the basal ganglia is the same; however, it is possible that HT reduces ongoing injury outside the primary lesion site. If so, delayed cooling still has a protective effect and longer cooling may be more effective in reducing secondary brain injury after ICH. Further studies should determine whether the protection is permanent or not. Second, BH treatment improves the forelimb placing test results, but not the corner turn test results 48 hours after ICH induction. This may reflect a difference in test sensitivity.29 The two tests used in this study are sensitive to detecting behavioral deficits in the rodent model of ICH.29 However, a previous study showed that forelimb placing test can detect even mild neurologic deficits such as those found after 15 minutes of the middle cere-
bral artery occlusion with reperfusion.30 A close temporal relationship between the brain edema and forelimb placing score is demonstrated in the rat ICH model.29 Third, we did not determine an exact time window of BH in this rat ICH model. When we started this study, we set the latest time point for 24 hours after ICH induction because later cooling would miss counteracting the early mechanism of brain injuries after ICH. However, there were no significant differences in the extent of suppression of the brain edema formation regardless of the timing of starting the HT therapy. We suspect that BH (even when delayed for 24 hours) counteracts multiple mechanisms of hemorrhagic tissue damage continuously and simultaneously (e.g., inflammation, oxidative stress, BBB disruption). Future study is necessary to answer this question whether delayed BH (after 2-3 days) can reduce perihematomal brain edema occurring in the subacute phase after ICH using an iron-infused brain injury model. In conclusion, mild BH significantly reduces brain edema formation after ICH, even when the BH is applied 24 hours after hematoma induction in rats. Several neuroprotective mechanisms including reduced BBB disruption, inflammation, and oxidative damage are suggested in this study. The treatment window of BH after ICH may be less constrained, probably indicating high applicability in clinical practice.
References 1. Xi G, Keep RF, Hoff JT. Mechanisms of brain injury after intracerebral hemorrhage. Lancet Neurol 2006;5:53-63. 2. Mendelow AD, Bullock R, Teasdale GM, et al. Intracranial hemorrhage induced at arterial pressure in the rat, part 2: Short-term changes in local cerebral blood flow measured by autoradiography. Neurol Res 1984; 6:189-193. 3. Yang GY, Betz AL, Chenevert TL, et al. Experimental intracerebral hemorrhage: Relationship between brain edema, blood flow, and blood-brain barrier permeability in rats. J Neurosurg 1994;81:93-102. 4. Lee KR, Colon GP, Betz AL, et al. Edema from intracerebral hemorrhage: The role of thrombin. J Neurosurg 1996; 84:91-96. 5. Huang F, Xi G, Keep RF, et al. Brain edema after experimental intracerebral hemorrhage: Role of hemoglobin degradation products. J Neurosurg 2002;96:287-293. 6. Hua Y, Keep RF, Hoff JT, et al. Brain injury after intracerebral hemorrhage: The role of thrombin and iron. Stroke 2007;38:759-762. 7. Wagner KR, Sharp FR, Ardizzone TD, et al. Heme and iron metabolism: Role in cerebral hemorrhage. J Cereb Blood Flow Metab 2003;23:629-652. 8. Wu J, Hua Y, Keep RF, et al. Oxidative brain injury from extravasated erythrocytes after intracerebral hemorrhage. Brain Res 2002;953:45-52. 9. Wu J, Hua Y, Keep RF, et al. Iron and iron-handing proteins in the brain after intracerebral hemorrhage. Stroke 2003;34:2964-2969.
DELAYED BRAIN HYPOTHERMIA FOR INTRACEREBRAL HEMORRHAGE 10. Gong C, Hoff JT, Keep RF. Acute inflammatory reaction following experimental intracerebral hemorrhage in rat. Brain Res 2000;871:57-65. 11. Hua Y, Xi G, Keep RF, et al. Complement activation in the brain after experimental intracerebral hemorrhage. J Neurosurg 2000;92:1016-1022. 12. Bernard S, Gray T, Buist M, et al. Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia. N Engl J Med 2002;346:557-563. 13. The Hypothermia after Cardiac Arrest Study Group. Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest. N Engl J Med 2002; 346:549-556. 14. Kawai N, Kawanishi M, Okauchi M, et al. Effects of hypothermia on thrombin-induced brain edema formation. Brain Res 2001;895:50-58. 15. MacLellan CL, Davies LM, Fingas MS, et al. The influence of hypothermia on outcome after intracerebral hemorrhage in rats. Stroke 2006;37:1266-1270. 16. Bradley BP, Priebat DA, Christensen RD, et al. Measurement of cutaneous inflammation: Estimation of neutrophils content with an enzyme marker. J Invest Dermatol 1982;78:206-209. 17. Kasai H, Crain PF, Kuchino Y, et al. Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair. Carcinogenesis 1986;7:1849-1851. 18. Gebel JM Jr, Jauch EC, Brott TG, et al. Natural history of perihematomal edema in patients with hyperacute spontaneous intracerebral hemorrhage. Stroke 2002;33: 2631-2635. 19. Lee KR, Kawai N, Kim S, et al. Mechanisms of edema formation after intracerebral hemorrhage: Effects of thrombin on cerebral blood flow, blood-brain barrier permeability, and cell survival in a rat model. J Neurosurg 1997;86:272-278.
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20. Xue M, Del Bigio MR. Acute tissue damage after injections of thrombin and plasmin into rat. Stroke 2001; 32:2164-2169. 21. Hua Y, Wu J, Keep RF, et al. Tumor necrosis factor-a increases in the brain after intracerebral hemorrhage and thrombin stimulation. Neurosurgery 2006;58:542-550. 22. Nakamura T, Keep RF, Hua Y, et al. Deferoxamineinduced attenuation of brain edema and neurological deficits in a rat model of intracerebral hemorrhage. J Neurosurg 2004;100:672-678. 23. Nakamura T, Keep RF, Hua Y, et al. Oxidative DNA injury after experimental intracerebral hemorrhage. Brain Res 2005;1039:30-36. 24. MacLellan CL, Girgis J, Colbourne F. Delayed onset of prolonged hypothermia improves outcome after intracerebral hemorrhage in rats. J Cereb Blood Flow Metab 2004;24:432-440. 25. Yanamoto H, Hong SC, Soleau S, et al. Mild postischemic hypothermia limits cerebral injury following transient focal ischemia in rat neocortex. Brain Res 1996;718:207-211. 26. Markgraf CG, Clifton GL, Moody MR. Treatment window for hypothermia in brain injury. J Neurosurg 2001; 95:979-983. 27. Smith SL, Hall ED. Mild pre- and posttraumatic hypothermia attenuates blood-brain barrier damage following controlled cortical impact injury in rat. J Neurotrauma 1996;13:1-9. 28. Inamasu J, Suga S, Sato S, et al. Post-ischemic hypothermia delayed neutrophil accumulation and microglial activation following transient focal ischemia in rats. J Neuroimmunol 1994;80:51-57. 29. Hua Y, Schallert T, Keep RF, et al. Behavioral tests after intracerebral hemorrhage in the rat. Stroke 2002; 33:2478-2484. 30. Hua Y, Wu J, Pecina S, et al. Ischemic preconditioning procedure induces behavioral deficits in the absence of brain injury. Neurol Res 2005;27:261-267.
Secondary consequences of intracerebral hemorrhage (ICH) including inflammation, edema, and oxidative damage all contribute to cell death after ICH. Brain hypothermia (BH) has been used as an effective neuroprotective treatment in experimental brain ischemia and traumatic brain injury. In this study, we first attempted to evaluate the effect of delayed mild BH (35 C) on brain edema formation 48 hours after ICH. BH was started 3, 6, 12, and 24 hours after the induction of 100 mL of autologous blood into the basal ganglia (hypothermic [HT]; HT3: n 5 4, HT6: n 5 6, HT12: n 5 11, HT24: n 5 6) in rats. To examine the protective mechanism of BH, blood-brain barrier (BBB) permeability to Evans blue, accumulation of polymorphonuclear leukocyte, and oxidative DNA damage in the lesion were compared between normothermic (NT) (37 C) and HT6 rats 48 hours after ICH. Finally, neurologic recovery was assessed using behavioral tests in NT and HT6 rats 48 hours after ICH. Brain water content in the ispilateral basal ganglia was significantly reduced with delayed BT compared with NT (n 5 7, 81.8 6 0.7% v HT3: 78.9 6 0.8%, P , .01; HT6: 78.7 6 0.6%, P , .01; HT12: 79.4 6 1.1%, P , .01; HT24: 80.3 6 0.6%, P , .01). The BBB disruption to Evans blue was significantly reduced with BH (HT6: n 5 6) compared with NT (n 5 6) rats in the ipsilateral basal ganglia (23.0 6 5.2 v 42.3 6 4.0 ng/g wet tissue, P , .05). HT6 treatment (n 5 6) significantly inhibited the accumulation of polymorphonuclear leukocyte compared with NT treatment (n 5 6) (0.43 6 0.22 v 1.49 6 0.61 DAbs/mg tissue, P ,.05). HT6 treatment (n 5 3) also significantly reduced oxidative DNA damage determined with 8-hydroxyl-2’-deoxyguanosine compared with NT treatment (n 5 3) (92 6 18 v 40 6 7 pg 8-hydroxyl-2’-deoxyguanosine/mg DNA, P , .05). Furthermore, HT6 treatment (n 5 5) significantly improved neurologic recovery assessed with forelimb placing score compared with NT treatment (42.0 6 5.8 v 12.0 6 3.7, P , .05). In conclusion, mild BH significantly reduces the brain edema formation after ICH, even when the BH is applied 24 hours after hematoma induction in rats. Several neuroprotective mechanisms, including reduced BBB disruption, inflammation and oxidative damage, are suggested in this study. Key Words: Blood-brain barrier—brain edema—brain hypothermia—intracerebral hemorrhage—oxidative damage—polymorphonuclear leukocyte. Ó 2008 by National Stroke Association
From the Departments of *Neurological Surgery and †Neurobiology, Kagawa University School of Medicine, Kagawa, Japan. Received October 12, 2007; revision received January 7, 2008; accepted January 30, 2008. Address correspondence to Nobuyuki Kawai, MD, Department of Neurological Surgery, Kagawa University School of Medicine, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. E-mail: nobu@med. kagawa-u.ac.jp. 1052-3057/$—see front matter Ó 2008 by National Stroke Association doi:10.1016/j.jstrokecerebrovasdis.2008.01.003
Spontaneous intracerebral hemorrhage (ICH) causes high mortality and poor neurologic recovery even in survivors. In ICH, significant tissue damage occurs immediately because of space-occupying effects and secondary consequences including brain edema formation contribute further tissue damage to cell death. Patients with ICH deteriorate progressively as a result of ongoing brain edema. A number of mechanisms may be involved in the brain edema formation after ICH.1 Brain edema after ICH
Journal of Stroke and Cerebrovascular Diseases, Vol. 17, No. 4 (July-August), 2008: pp 187-195
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was believed to be caused by local pressure compressing the microcirculation and producing ischemia in the region around the hematoma.2 However, there is no experimental evidence that the degree or duration of hypoperfusion around the hematoma is sufficient to produce ischemic brain damage.3 Attention later focused on the direct role of the biochemical substances from an intracerebral blood clot in generating brain edema. At least two phases of edema formation are involved in ICH.1 These include an acute phase (first 2 days) involving the activation of the coagulation cascade and thrombin formation and a subacute phase (after 3 days) involving red blood cell lysis and hemoglobin-induced neuronal toxicity.4-6 Iron is the hemoglobin degradation product and iron overload in the brain can cause free radical formation and oxidative damage such as lipid peroxidation after ICH.7-9 Inflammatory reactions including leukocyte accumulation and activation of the complimentary system in the brain parenchyma also play an important role in ICH-related brain edema formation.10,11 Mild, prolonged brain hypothermia (BH) improves outcome in rodent models of global and focal ischemia. Clinically, BH can be safely applied to patients with severe head trauma and stroke. Recently, renewed interest has been directed to global ischemia in resuscitated patients with cardiac arrest and clinical trials report improved outcome when prolonged BH is induced soon after cardiac arrest.12,13 BH may reduce secondary consequences of ICH such as edema, blood-brain barrier (BBB) disruption, inflammation, and oxidative damage in the perihematomal region. Early work from our laboratory shows that immediate BH reduces vasogenic edema formation after intrastriatal thrombin injection in rats.14 Recently, MacLellan et al15 revealed that BH initiated 1 or 4 hours after autologous blood infusion into the striatum reduces brain edema formation, BBB disruption, and inflammatory cell infiltration. We hypothesized that delayed BH would improve outcome in the whole blood model of ICH because progression of brain injury around hematoma is an ongoing phenomenon.1 In this study, we assessed whether delayed post-ICH BH affects the brain edema formation, BBB permeability, inflammation, oxidative damage, and behavioral recovery.
Materials and Methods The experimental protocols used in this study were approved by our ethics committees for animal experiment. Animals were allowed free access to food and water before the experiment.
(60 mg/kg). A polyethylene (PE-50) catheter was inserted into the femoral artery to monitor the arterial blood pressure and to obtain arterial blood for analysis of blood gases, blood pH, hematocrit, and blood glucose. The rat’s body (rectal) temperature was maintained at 37 C during the surgery using a feedback-controlled heating system. The rats were positioned in a stereotactic frame and the scalp was incised along the midline. Using a sterile technique, a 1 mm burr hole was opened in the skull on the left coronal suture 3 mm lateral to the midline. A blunt 26-gauge needle was inserted into the left basal ganglia under stereotactic guidance (coordinates: 0.2 mm anterior, 6.0 mm ventral, and 3.0 mm lateral to the bregma). Then, a 100 mL of autologous whole blood was infused at a rate of 20 mL/min with the use of a microinfusion pump (EPS-26, Eicom, Kyoto, Japan). After completion of the infusion, the needle was withdrawn quickly and cyanoacrylate glue was placed around the burr hole, and the skin incision was closed with sutures. After the surgery, rats were divided into normothermic (NT) and hypothermic (HT) groups. NT rats were housed in a room maintained at 25 C for 48 hours and HT rats were housed in a cold room maintained at 5 C with fine water misters for cooling. HT treatment was started 3 (HT3), 6 (HT6), 12 (HT12), and 24 (HT24) hours after the induction of ICH. In a pilot study, we examined what degree of brain temperature was produced in a room maintained at 25 C (n 5 5) and in a cold room maintained at 5 C (n 5 5) in awake rats using a telemetry system. A needle-type thermotelemeter (TA10TA-F20, Data Sciences, St. Paul, Minn) was inserted into the brain at a 6-mm depth on the coronal suture. The data from the thermotelemeter were sent to the telemetry receiver (RPC-1, Data Sciences) and continuously monitored for 48 hours.
Experimental Protocols This study was divided into 3 parts. The first part evaluated the effect of brain temperature on the brain edema formation 48 hours after ICH in NT (n 5 7) and HT (HT3: n 5 4, HT6: n 5 6, HT12: n 5 11, HT24: n 5 6) rats. In the second part, the BBB permeability to Evans blue (EB) dye, accumulation of the polymorphonuclear (PMN) leukocyte, and oxidative DNA damage in the lesion 48 hours after ICH were examined in NT and HT6 rats to clarify the protective mechanism of BH. In this part, 6 rats were used for EB and PMN studies and 3 rats were used to study oxidative damage in each group. Finally, neurologic recovery was assessed using two behavioral tests in NT (n 5 5) and HT6 (n 5 5) rats 24 and 48 hours after ICH.
Animal Preparation and Intracerebral Blood Infusion Adult male Sprague-Dawley rats (Charles River Laboratories, Hino, Japan) weighing 250 to 400 g were anesthetized with intraperitoneal sodium pentobarbital
Measurement of Brain Water Content The rats were killed by decapitation under deep pentobarbital anesthesia 48 hours after ICH induction. The
DELAYED BRAIN HYPOTHERMIA FOR INTRACEREBRAL HEMORRHAGE
brains were removed rapidly and two coronal slices 3 mm from the frontal pole were cut 3-mm thick. The anterior slice (slice A) contained the needle trajectory and arterial blood injection site at the basal ganglia. The adjacent slice (slice B) was a section posterior to the arterial blood injection site. These sections of brain were divided along the midline and the cortex was separated from the basal ganglia bilaterally. The hematoma itself in the basal ganglia was removed gently from the surrounding tissue. The tissue samples were immediately weighed on an electronic analytic balance to the nearest 0.1 mg to obtain the wet weight (WW). The tissue was then dried in an oven at 110 C for 24 hours and weighed again to obtain the dry weight. The formula, (WW – dry weight)/WW 3 100, was used to calculated the water content and expressed as percentage WW.
BBB Permeability to EB BBB integrity was investigated using EB dye extravasation. EB solution (Sigma Chemical Co, St Louis, Mo) (2% in saline; 2 mL/kg) was injected intravenously 48 hours after ICH induction and allowed to circulate for 30 minutes under sodium pentobarbital anesthesia (60 mg/kg). The chest was subsequently opened and the brain was transcardially perfused with 200 mL of saline through the left ventricle at 100-mm Hg pressure until almost colorless perfusion fluid was obtained from the right atrium. After decapitation, the brains were removed and two coronal slices (slices A and B) 3 mm from the anterior pole were cut 3-mm thick. These sections of the brain were divided along the midline and the cortex was separated from the basal ganglia. The hematoma in the basal ganglia was removed from the surrounding tissue. Samples were then weighed and placed in 50% trichloroacetic solution. After homogenization and centrifugation, the extracted dye was diluted with ethanol (1:3), and its fluorescence was determined (excitation at 620 nm and emission at 680 nm) with a luminescence spectrophotometer (65040, Hitachi, Tokyo, Japan). Calculations were based on an external standard (62.5-500 ng/mL) in the same solvent. The tissue content of EB was quantified from a linear standard line and was expressed per gram of tissue.
Measurement of Myeloperoxidase Activity Myeloperoxidase (MPO) activity was used as an indicator of tissue PMN leukocyte accumulation. The brains were transcardially perfused with 200 mL of cold saline through the left ventricle at 100-mm Hg pressure and removed rapidly. A coronal slice 3 mm from the frontal pole was cut 5-mm thick and separated into right and left hemispheres. The hematoma was removed from the basal ganglia. Each hemisphere was immediately frozen in liquid nitrogen and stored at –80 C for later analysis. The procedure described by Bradley et al16 was used for the quantification of MPO activity with minor modification.
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The brain samples were thawed on ice and the cortex was separated from the basal ganglia. The WW of each sample was immediately measured. Each sample was homogenized in 5-mmol/L potassium phosphate buffer (pH 6.0, 4 C) at an original tissue weight:volume ratio of 1:20 and centrifuged at 30,000 3 g (30 minutes, 4 C). The supernatant was discarded and the pellet was washed again as described above. After decanting the second supernatant, the pellet was extracted by suspension in 0.5 hexadecyltrimethylammonium bromide in 50 mmol/L potassium phosphate buffer (pH 6.0, 25 C) for approximately 2 minutes at an original tissue weight:volume ratio of 1:10. The samples were immediately frozen in liquid nitrogen. Three freeze/thaw cycles were performed with sonications (10 seconds, 25 C) between cycles. After the last sonication, the samples were incubated at 4 C for 20 minutes and centrifuged at 12,500 3 g (15 minutes, 4 C). A 0.1-mL aliquot of supernatant was mixed with 2.9 mL of 50-mmol/L potassium phosphate buffer, pH 6.0, containing 0.167 mg/mL of o-dianisidine dihydrochloride and 0.005% hydrogen peroxide. The change in absorbance at 460 nm during 2 minutes was measured by a spectrophotometer (U-3300, Hitachi). All chemicals used in this study were supplied by Sigma Chemical Co.
Measurement of 8-Hydroxyl-2’-Deoxyguanosine in DNA DNA is vulnerable to oxidative stress. Deoxyguanosine residues in DNA are hydroxylated by various agents that produce free radicals resulting in 8-hydroxyl-2’-deoxyguanosine (8-OHdG) accumulations. 8-OHdG might serve as a sensitive biomarker of intracellular oxidative stress.17 Brains were treated rapidly and stored as described in the MPO study. DNA extraction was performed using a DNA isolation kit (Dojindo Molecular Technologies, Kumamoto, Japan). The levels of 8-OHdG of the samples were determined using enzyme-linked immunosorbent assay kit (Japan Institute for the Control of Aging, Shizuoka, Japan). The kit measures extremely low levels of 8-OHdG, and the specificity of the monoclonal antibody has been established. The wells were subjected to optical density measurement at 450 nm. 8-OHdG enzyme-linked immunosorbent assay was triplicate and the means were calculated. The data, expressed as pg 8-OHdG/mg DNA, were calculated based on the linear calibration curve generated for each experiment using 8-OHdG standard solutions.
Behavioral Tests (Forelimb Placing Test and Corner Turn Test) Forelimb placing was scored using the vibrissae-elicited forelimb placing test. Animals were held by their bodies to allow their forelimbs to hang free. Independent testing of each forelimb was induced by brushing the
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Table 1. Physiologic parameters
No. of animals MABP (mm Hg) pH PaCO2 (mm Hg) PaO2 (mm Hg) Hematocrit (%) Blood glucose (mg/dL)
NT
HT3
HT6
HT12
HT24
7 114 6 8 7.42 6 0.05 42 6 8 96 6 11 42 6 4 106 6 19
6 108 6 10 7.43 6 0.06 44 6 6 85 6 9 44 6 5 99 6 16
6 111 6 6 7.44 6 0.04 43 6 4 89 6 7 44 6 6 103 6 21
11 102 6 7 7.47 6 0.06 41 6 5 94 6 14 43 6 5 103 6 15
6 103 6 8 7.41 6 0.05 45 6 5 84 6 8 44 6 4 97 6 20
Abbreviations: HT, hypothermia treatment started 3 (HT3), 6 (HT6), 12 (HT12), and 24 (HT24) hours after induction of intracerebral hemorrhage; MABP, mean arterial blood pressure; NT, normothermia. Measurements were performed in each group at the time of autologous blood injection. Values are expressed as mean 6 SD.
Statistical Analysis Differences in the brain edema formation among groups (NT, HT3, HT6, HT12, and HT24) were analyzed using analysis of variance and the Scheffe´ F test of significance. The physiologic variables, EB extravasations, MPO activities, 8-OHdG levels, and behavioral scores (corner turn score and forelimb placing score) in the NT and HT6 groups were compared by unpaired Student’s t test. A two-tailed probability value of less than .05 was used to indicate a significant difference. All values are expressed as mean 6 SEM.
Results Physiologic Parameters Table 1 shows the mean values of physiologic parameters in NT and HT (HT3, HT6, HT12, and HT24) rats used in the brain water content study. There were no significant differences in the values of arterial blood pressure, blood gas tensions, hematocrit level, and blood glucose concentration at the time of autologous blood injection among the groups. According to the pilot study, the brain temperature in rats housed in a cold room maintained at 5 C reduced
temporarily to about 30 C and then gradually recovered to 35 C, which lasted throughout the observation (Fig 1).
Brain Water Content Intracerebral injection of a 100-mL autologous blood produced medium ICH in the basal ganglia, which sometimes extended to the lateral ventricle (Fig 2, A). BH treatment significantly reduced the brain edema formation in the ipsilateral basal ganglia (NT: 81.8 6 0.7% v HT3: 78.9 6 0.8%, P , .01; HT6: 78.7 6 0.6%, P , .01; HT12: 79.4 6 1.1%, P , .01; HT24: 80.3 6 0.6%, P , .01) (Fig 3, A) and cortex (NT: 81.0 6 1.0% v HT3: 79.6 6 0.9%, P , .05; HT6: 79.1 6 0.9%, P , .01; HT12: 79.3 6 0.8%, P , .01; HT24: 79.5 6 0.3%, P , .01) (Fig 3, B) in the slice of blood injection (slice A). An increase in brain water content was also significantly suppressed with BH in the ipsilateral basal ganglia (NT: 80.9 6 0.9% v HT3: 78.5 6 0.3%, P , .01; HT6: 78.9 6 0.4%, P , .01; HT12: 79.5 6 0.9%, P , .01; HT24: 79.7 6 0.8%, P , .05) (Fig 3, C) and cortex (NT: 79.5 6 0.9% v HT3: 78.6 6 0.6%, P , .05; HT6: 78.6 6 0.4%, P , .05; HT12: 78.8 6 0.8%, P , .05; HT24: 78.8 6 0.6%, P , .05) (Fig 3, D) in the slice adjacent 45
Brain temperature (°C)
respective vibrissae on the corner of a table top once per trial for 10 trials. A score of one was given each time the rat placed its forelimb onto the edge of the table in response to the vibrissae stimulation. Percentage success in placing responses was determined for impaired forelimb and nonimpaired forelimb. Corner turn test was also used in this study. For the corner turn test, the rat was allowed to proceed into a corner, the angle of which was 30 degrees. To exit the corner, the animal could turn either to the left or right, and this was recorded. This was repeated 10 to 15 times, and the percentage of right turns was calculated. Both forelimb placing and corner turn tests were performed and scored by an investigator blind to the treatment conditions.
Normo Hypo 40
35
30
25
0
4
8
12
16
20
24
28
32
36
40
44
48
Time course (hours) Figure 1. Line graphs depicting time-course changes in brain temperature in rats housed in room maintained at 25 C (normo) (n 5 5) and in cold room maintained at 5 C (hypo) (n 5 5). Measurements were made in awake rats using telemetry system for 24 hours.
DELAYED BRAIN HYPOTHERMIA FOR INTRACEREBRAL HEMORRHAGE
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Figure 2. (A) Photographs showing coronal sections (2-mm thick) containing hematoma 24 hours after autologous blood infusion. (B) Photographs showing Evans blue staining observed in basal ganglia adjacent to hematoma (small arrows) and overlying ipsilateral cortex (large arrows).
to blood injection (slice B). Differences in the brain water contents were not statistically significant among the HT subgroups.
BBB Permeability to EB As shown in Fig 4, EB staining was observed in the basal ganglia adjacent to the hematoma and overlying ipsilateral cortex, indicating increased BBB permeability and vasogenic edema development (Fig 2, B). The BBB was significantly protected from breakdown with BH compared with NT rat in the ispilateral basal ganglia (NT: 42.3 6 4.0 v HT6: 23.0 6 5.2 ng/g wet tissue, P , .05), but not in the ipsilateral cortex (NT: 53.3 6 10.6 v HT6: 33.8 6 10.4 ng/g wet tissue) in the slice of blood injection (slice A). In the adjacent slice B, the BBB disruption was significantly inhibited with BH compared with NT in the ipsilateral basal ganglia (NT: 55.5 6 9.3 v HT6: 21.8 6 3.2 ng/g wet tissue, P , .05) and cortex (NT: 34.9 6 9.3 v HT6: 16.6 6 3.5 ng/g wet tissue, P , .01).
MPO Activity As shown in Fig 5, A, using light microscopy, a prominent perihematomal accumulation of PMN leukocytes was observed in NT rats 48 hours after ICH induction. The MPO activity is used as an indicator of tissue PMN leukocyte accumulation. There was a significant effect with BH on tissue MPO activity. BT significantly reduced the accumulation of PMN leukocytes in the ipsilateral hemisphere compared with NT rats (NT: 1.49 6 0.61 v HT6: 0.43 6 0.22 DAbs/mg tissue, P , .05). A significant difference was not observed in the contralateral hemisphere between the NT and HT6 groups.
8-OHdG in DNA As shown in Fig 5, B, 8-OHdG is formed from deoxyguanosine in DNA by hydroxyl free radicals and might serve as a sensitive biomarker of intracellular oxidative stress. 8-OHdG level in the extracted DNA is significantly lower in the ipsilateral hemisphere treated with BH
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A85
B 85 NT HT3
83
HT6
81 79
**
**
HT12 HT24
** **
77 75
HT12
** ** **
HT24
79 77
Ipsilateral
Contralateral
D 85 HT6
81
** ** **
*
Cortex: slice B
NT HT3
83
HT12 HT24
77 75
Brain Water Content (%)
BG: slice B
Brain Water Content (%)
HT6
*
81
Contralateral
85
79
HT3
83
75
Ipsilateral
C
NT
Cortex: slice A
Brain Water Content (%)
Brain Water Content (%)
BG: slice A
NT HT3
83
HT6
81
HT12
*
79
*
HT24
* *
Figure 3. Bar graph comparing brain water content between NT (n 5 7) and HT (HT3: n 5 4, HT6: n 5 6, HT12: n 5 11, HT24: n 5 6) rats in slice of hematoma injection ([A] basal ganglia [BG], [B] cortex) and in adjacent slice ([C] BG, [D] cortex) 48 hours after ICH. Hypothermia significantly reduced brain water content in ipsilateral BG and cortex in slice of hematoma injection compared with NT rats. In adjacent slice, HT also significantly reduced brain water content in ipsilateral BG and cortex in slice of hematoma injection. Differences in brain water content were not significant among HT subgroups (HT3, HT6, HT12, and HT24). **P , .01 and *P , .05.
77 75
Ipsilateral
Contralateral
Ipsilateral
compared with that in NT rats (NT: 92 6 18 v HT6: 40 6 7 pg/mg DNA, P , .05). A significant difference was not observed in the contralateral hemisphere between the NT and HT6 groups.
Contralateral
when the hematoma mass initially compressed the brain resulting in microcirculatory disorder with consequent ischemia in the brain tissue surrounding the hematoma.2 However, there is no experimental evidence that the degree or duration of hypoperfusion around the hematoma
Behavioral Tests
Discussion ICH is a common emergency stroke treated by neurologists and neurosurgeons, and its neurologic manifestations may frequently exacerbate as a result of brain edema after initial brain damage with hematoma. Brain edema after ICH gradually develops within 24 hours after the ictus and continues to develop for the next couple of days.18 The underlying mechanism of brain edema formation after ICH is complex and has not been fully elucidated.1 Brain edema after ICH was believed to occur
70 NT
BBB Permeability to Evan’s Blue (ng / g wet tissue)
As shown in Fig 6, neurologic deficits were examined using the forelimb placing test and corner turn test 24 and 48 hours after ICH. The forelimb placing score was significantly improved with BH initiated 6 hours after ICH compared with NT rats 48 hours after ICH (NT: 12 6 8 v HT6: 42 6 13%, P , .05), but not 24 hours after ICH (NT: 2 6 4 v HT6: 14 6 13%, P 5 .09). On the other hand, the corner turn score was not significantly different between the NT and HT6 groups examined at 24 and 48 hours after ICH induction.
60
HT6
50 40
*
30
* **
20 10 0 Cortex
BG
Slice A
BG
Cortex
Slice B
Figure 4. Bar graph comparing BBB permeability to EB between NT (n 5 6) and HT (HT6: n 5 6) rats in slice of hematoma injection (slice A) and in adjacent slice (slice B) 48 hours after ICH. Hypothermia significantly reduced BBB permeability to EB in ipsilateral basal ganglia (BG) in slice A and in ipsilateral BG and cortex in slice B compared with NT group. **P , .01 and *P , .05.
DELAYED BRAIN HYPOTHERMIA FOR INTRACEREBRAL HEMORRHAGE
A
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B
2.5
120
2
1.5
1
* 0.5
125 NT
Forelimb Placing Score (%)
60
* 40
0
is sufficient to produce ischemic brain damage.3 Several studies have demonstrated that toxic substances released from the clot are involved in accelerating the brain injury.4-6 These include the acute phase (first 2 days) involving the activation of the coagulation cascade and thrombin formation and the subacute phase (after 3 days) involving red blood cell lysis and hemoglobininduced neuronal toxicity. Thrombin is involved in the development of brain injury after ICH.4 Thrombin induces BBB disruption and the death of parenchymal cells directly.19 There is also evidence that thrombin has effects related to inflammation with chemotaxis of leukocytes, activation of microglia, and cytokine release.14,20,21 Red blood cell lysis and hemoglobin toxicity also lead to
HT6
75
* 50
25
0 24 hours
80
20
ipsilateral
Before
HT6
HT6
0
100
NT 100
8-OHdG (pg / µg DNA)
MPO Activity (
Figure 5. (A) Bar graph comparing MPO activity in ispilateral hemisphere between NT (n 5 6) and HT (HT6: n 5 6) rats 48 hours after ICH. HT treatment significantly reduced PMN leukocyte accumulation compared with NT. (B) Bar graph comparing 8-OHdG level in DNA extraction between NT (n 5 3) and HT (HT6: n 5 3) rats 48 hours after ICH. HT treatment significantly reduced 8-OHdG concentration (pg) per mg DNA in ipsilateral hemisphere compared with NT. (A and B) Significant difference was not observed in contralateral hemisphere between two groups. *P , .05.
Abs / mg tissue)
NT
48 hours
Figure 6. Bar graph comparing forelimb placing score between NT (n 5 5) and HT (HT6: n 5 5) rats 24 and 48 hours after ICH. Forelimb placing score was significantly improved with HT treatment compared with NT 48 hours after ICH, but not 24 hours after ICH. *P, .05.
contralateral
ipsilateral
contralateral
secondary injury after ICH.1,5,6 Iron is a hemoglobin degradation product and iron overload can lead to detrimental results on the brain tissue.7-9 A previous study showed that intracerebral infusion of iron causes brain edema, and that an iron chelator, deferoxamine, reduces hemoglobin-induced brain edema, indicating that iron plays an important role in edema formation after ICH.22 A recent study demonstrates that infusion of autologous whole blood and ferrous iron into the basal ganglia causes oxidative DNA damage, suggesting that iron might contribute to ICH-induced oxidative stress.23 Mild, prolonged BH improves functional recovery and reduces injury after global and focal ischemia in rodents. Mild BH is a safe and feasible treatment for head injury and stroke victims. Notably, recent clinical trials report improved outcome when prolonged BH is induced in resuscitated patients with cardiac arrest.12,13 A number of underlying mechanisms have been proposed in the neuroprotective effects of BH in experimental ischemic brain injury. Secondary consequences of ischemia and ICH include inflammation, edema, and oxidative stress, which all lead to cell death. Given the overlap in mechanisms contributing to injury after ischemia and ICH, it makes sense to apply BH to ICH. Our previous study revealed that early BH reduces the BBB disruption, PMN leukocyte accumulation, and brain edema after intrastriatal thrombin injection in rats.14 Unexpectedly, however, MacLellan et al15 demonstrated that BH initiated soon after collagenase-induced ICH fails to reduce tissue damage or behavioral deficits. This result is attributable to general side effects of BH (e.g., elevated blood pressure, coagulopathy), which would counteract beneficial effects of BH during the early post-ICH period. Later, they showed that BH delayed 12 hours after collagenase-induced ICH improves recovery and lessens tissue loss because
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bleeding would not be aggravated at this time. Recently, MacLellan et al15 revealed that BH initiated 1 or 4 hours after autologous blood infusion into the striatum reduces brain edema formation, BBB disruption, and inflammatory cell infiltration. We hypothesized that delayed BH would improve outcome in the whole blood ICH model because progression of brain injuries around hematoma is an ongoing phenomenon. In this study, we clearly demonstrate that delayed BH initiated 3 to 24 hours after ICH induction exhibits protective effect in rats. Our study also revealed that delayed BH initiated 6 hours after ICH reduced PMN leukocyte accumulation and oxidative DNA damage in the lesion. These are not a result of general physiologic effects of BH on the brain because a significant difference was not observed in the contralateral hemisphere between NT and HT rats, indicating that HT per se does not influence the PMN infiltration and oxidative DNA damage in the normal brain tissue. Previous studies have shown that the treatment time window for BH in experimental ischemic and traumatic brain injuries is narrow.25,26 In this model of ICH, an early study demonstrated that the brain edema in the basal ganglia develops progressively over 24 hours and remains relatively constant for 4 days before beginning to resolve.3 It is reported that BH can promptly reduce BBB breakdown after traumatic brain injury in rats.27 Therefore, BH applied 24 hours after ICH induction could improve perihematomal BBB damage and reduce vasogenic brain edema in the basal ganglia. There are some limitations in this study. First, we did not determine whether delayed BH provides long-lasting neuroprotection in rats with ICH. BH may have postponed harmful processes around hematoma as found in cerebral ischemia, in which BH sometimes delays inflammation and neuronal damage.28 A previous study demonstrated that BH started 12 hours after collagenase injection into the basal ganglia reduces chronic tissue loss 30 days after ICH.24 However, a recent study shows that BH started 1 and 4 hours after autologous whole blood infusion into the striatum does not affect lesion volume 30 days after ICH induction.15 In the whole blood infusion ICH model, the initial volume of tissue damage in the basal ganglia is the same; however, it is possible that HT reduces ongoing injury outside the primary lesion site. If so, delayed cooling still has a protective effect and longer cooling may be more effective in reducing secondary brain injury after ICH. Further studies should determine whether the protection is permanent or not. Second, BH treatment improves the forelimb placing test results, but not the corner turn test results 48 hours after ICH induction. This may reflect a difference in test sensitivity.29 The two tests used in this study are sensitive to detecting behavioral deficits in the rodent model of ICH.29 However, a previous study showed that forelimb placing test can detect even mild neurologic deficits such as those found after 15 minutes of the middle cere-
bral artery occlusion with reperfusion.30 A close temporal relationship between the brain edema and forelimb placing score is demonstrated in the rat ICH model.29 Third, we did not determine an exact time window of BH in this rat ICH model. When we started this study, we set the latest time point for 24 hours after ICH induction because later cooling would miss counteracting the early mechanism of brain injuries after ICH. However, there were no significant differences in the extent of suppression of the brain edema formation regardless of the timing of starting the HT therapy. We suspect that BH (even when delayed for 24 hours) counteracts multiple mechanisms of hemorrhagic tissue damage continuously and simultaneously (e.g., inflammation, oxidative stress, BBB disruption). Future study is necessary to answer this question whether delayed BH (after 2-3 days) can reduce perihematomal brain edema occurring in the subacute phase after ICH using an iron-infused brain injury model. In conclusion, mild BH significantly reduces brain edema formation after ICH, even when the BH is applied 24 hours after hematoma induction in rats. Several neuroprotective mechanisms including reduced BBB disruption, inflammation, and oxidative damage are suggested in this study. The treatment window of BH after ICH may be less constrained, probably indicating high applicability in clinical practice.
References 1. Xi G, Keep RF, Hoff JT. Mechanisms of brain injury after intracerebral hemorrhage. Lancet Neurol 2006;5:53-63. 2. Mendelow AD, Bullock R, Teasdale GM, et al. Intracranial hemorrhage induced at arterial pressure in the rat, part 2: Short-term changes in local cerebral blood flow measured by autoradiography. Neurol Res 1984; 6:189-193. 3. Yang GY, Betz AL, Chenevert TL, et al. Experimental intracerebral hemorrhage: Relationship between brain edema, blood flow, and blood-brain barrier permeability in rats. J Neurosurg 1994;81:93-102. 4. Lee KR, Colon GP, Betz AL, et al. Edema from intracerebral hemorrhage: The role of thrombin. J Neurosurg 1996; 84:91-96. 5. Huang F, Xi G, Keep RF, et al. Brain edema after experimental intracerebral hemorrhage: Role of hemoglobin degradation products. J Neurosurg 2002;96:287-293. 6. Hua Y, Keep RF, Hoff JT, et al. Brain injury after intracerebral hemorrhage: The role of thrombin and iron. Stroke 2007;38:759-762. 7. Wagner KR, Sharp FR, Ardizzone TD, et al. Heme and iron metabolism: Role in cerebral hemorrhage. J Cereb Blood Flow Metab 2003;23:629-652. 8. Wu J, Hua Y, Keep RF, et al. Oxidative brain injury from extravasated erythrocytes after intracerebral hemorrhage. Brain Res 2002;953:45-52. 9. Wu J, Hua Y, Keep RF, et al. Iron and iron-handing proteins in the brain after intracerebral hemorrhage. Stroke 2003;34:2964-2969.
DELAYED BRAIN HYPOTHERMIA FOR INTRACEREBRAL HEMORRHAGE 10. Gong C, Hoff JT, Keep RF. Acute inflammatory reaction following experimental intracerebral hemorrhage in rat. Brain Res 2000;871:57-65. 11. Hua Y, Xi G, Keep RF, et al. Complement activation in the brain after experimental intracerebral hemorrhage. J Neurosurg 2000;92:1016-1022. 12. Bernard S, Gray T, Buist M, et al. Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia. N Engl J Med 2002;346:557-563. 13. The Hypothermia after Cardiac Arrest Study Group. Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest. N Engl J Med 2002; 346:549-556. 14. Kawai N, Kawanishi M, Okauchi M, et al. Effects of hypothermia on thrombin-induced brain edema formation. Brain Res 2001;895:50-58. 15. MacLellan CL, Davies LM, Fingas MS, et al. The influence of hypothermia on outcome after intracerebral hemorrhage in rats. Stroke 2006;37:1266-1270. 16. Bradley BP, Priebat DA, Christensen RD, et al. Measurement of cutaneous inflammation: Estimation of neutrophils content with an enzyme marker. J Invest Dermatol 1982;78:206-209. 17. Kasai H, Crain PF, Kuchino Y, et al. Formation of 8-hydroxyguanine moiety in cellular DNA by agents producing oxygen radicals and evidence for its repair. Carcinogenesis 1986;7:1849-1851. 18. Gebel JM Jr, Jauch EC, Brott TG, et al. Natural history of perihematomal edema in patients with hyperacute spontaneous intracerebral hemorrhage. Stroke 2002;33: 2631-2635. 19. Lee KR, Kawai N, Kim S, et al. Mechanisms of edema formation after intracerebral hemorrhage: Effects of thrombin on cerebral blood flow, blood-brain barrier permeability, and cell survival in a rat model. J Neurosurg 1997;86:272-278.
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20. Xue M, Del Bigio MR. Acute tissue damage after injections of thrombin and plasmin into rat. Stroke 2001; 32:2164-2169. 21. Hua Y, Wu J, Keep RF, et al. Tumor necrosis factor-a increases in the brain after intracerebral hemorrhage and thrombin stimulation. Neurosurgery 2006;58:542-550. 22. Nakamura T, Keep RF, Hua Y, et al. Deferoxamineinduced attenuation of brain edema and neurological deficits in a rat model of intracerebral hemorrhage. J Neurosurg 2004;100:672-678. 23. Nakamura T, Keep RF, Hua Y, et al. Oxidative DNA injury after experimental intracerebral hemorrhage. Brain Res 2005;1039:30-36. 24. MacLellan CL, Girgis J, Colbourne F. Delayed onset of prolonged hypothermia improves outcome after intracerebral hemorrhage in rats. J Cereb Blood Flow Metab 2004;24:432-440. 25. Yanamoto H, Hong SC, Soleau S, et al. Mild postischemic hypothermia limits cerebral injury following transient focal ischemia in rat neocortex. Brain Res 1996;718:207-211. 26. Markgraf CG, Clifton GL, Moody MR. Treatment window for hypothermia in brain injury. J Neurosurg 2001; 95:979-983. 27. Smith SL, Hall ED. Mild pre- and posttraumatic hypothermia attenuates blood-brain barrier damage following controlled cortical impact injury in rat. J Neurotrauma 1996;13:1-9. 28. Inamasu J, Suga S, Sato S, et al. Post-ischemic hypothermia delayed neutrophil accumulation and microglial activation following transient focal ischemia in rats. J Neuroimmunol 1994;80:51-57. 29. Hua Y, Schallert T, Keep RF, et al. Behavioral tests after intracerebral hemorrhage in the rat. Stroke 2002; 33:2478-2484. 30. Hua Y, Wu J, Pecina S, et al. Ischemic preconditioning procedure induces behavioral deficits in the absence of brain injury. Neurol Res 2005;27:261-267.