Early Transient Mild Hypothermia Attenuates Neurologic Deficits and Brain Damage After Experimental Subarachnoid Hemorrhage in Rats

Original Article

Early Transient Mild Hypothermia Attenuates Neurologic Deficits and Brain Damage After Experimental Subarachnoid Hemorrhage in Rats Nadine Lilla, Christoph Rinne, Judith Weiland, Thomas Linsenmann, Ralf-Ingo Ernestus, Thomas Westermaier

OBJECTIVE: Metabolic exhaustion in ischemic tissue is the basis for a detrimental cascade of cell damage. In the acute stage of subarachnoid hemorrhage (SAH), a sequence of global and focal ischemia occurs, threatening brain tissue to undergo ischemic damage. This study was conducted to investigate whether early therapy with moderate hypothermia can offer neuroprotection after experimental SAH.

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METHODS: Twenty male Sprague-Dawley rats were subjected to SAH and treated by active cooling (34
C) or served as controls by continuous maintenance of normothermia (37.0
C). Mean arterial blood pressure, intracranial pressure, and local cerebral blood flow over both hemispheres were continuously measured. Neurologic assessment was performed 24 hours later. Hippocampal damage was assessed by hematoxylin-eosin and caspase-3 staining.

slowing down metabolic exhaustion by hypothermia may still be a valuable treatment during this state of ischemic brain damage and prolong the therapeutic window for possible causal treatments of the acute perfusion deficit. Therefore, it may be useful as a first-tier therapy in suspected SAH.

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RESULTS: By a slight increase of mean arterial blood pressure in the cooling phase and a significant reduction of intracranial pressure, hypothermia improved cerebral perfusion pressure in the first 60 minutes after SAH. Accordingly, a trend to increased cerebral blood flow was observed during this period. The rate of injured neurons was significantly reduced in hypothermia-treated animals compared with normothermic controls.

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CONCLUSIONS: The results of this series cannot finally answer whether this form of treatment permanently attenuates or only delays ischemic damage. In the latter case,

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Key words Acute vasospasm - CBF - Hypothermia - Neuroprotection - Subarachnoid hemorrhage -

Abbreviations and Acronyms CBF: Cerebral blood flow CPP: Cerebral perfusion pressure HE: Hematoxylin-eosin ICA: Internal carotid artery ICP: Intracranial pressure LCBF: Local cerebral blood flow LDF: Laser-Doppler flowmeter

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INTRODUCTION

A

fter the onset of ischemia, energy failure results in the breakdown of membrane potentials and a loss of cellular electrolyte gradients and cellular compartmentation. Metabolic pathways stop working appropriately and the cell enters a detrimental cascade of ischemic damage. Therapeutic interventions that aim at preserving energy metabolism or at preventing the depletion of energy substrates may therefore offer tissue protection. The neuroprotective effect of hypothermia in ischemic conditions of the brain is undisputed. Deep hypothermia is therefore routinely used in cardiac surgery. During the last 3 decades, the neuroprotective properties of moderate or even mild hypothermia have been evaluated in various experimental models of traumatic and ischemic brain damage1-4 and are summarized in Table 1. Mild hypothermia has shown an extraordinarily high tissue protective effect in literally all experimental studies.15,20,32,35 However, the effect of mild hypothermia has not been reproducible in clinical studies of traumatic brain injury36 and is being tested in embolic stroke.37 The reason for its lack of efficiency in clinical trials may be that the stage of irreversible damage may largely have already been entered by the time the

MABP: Mean arterial blood pressure MCA: Middle cerebral artery PBS: Phosphate-buffered saline SAH: Subarachnoid hemorrhage Department of Neurosurgery, University Hospital Wuerzburg, Wuerzburg, Germany To whom correspondence should be addressed: Nadine Lilla, M.D. [E-mail: [email protected]] Citation: World Neurosurg. (2017). https://doi.org/10.1016/j.wneu.2017.09.109 Journal homepage: www.WORLDNEUROSURGERY.org Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2017 Elsevier Inc. All rights reserved.

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Table 1. Previous Studies on the Effects of Hypothermia on Cerebral Blood Flow in Experimental Models with and without Specific Brain Lesions Experimental Model of Brain Ischemia/Trauma

Reference

Temperature

Species

Buckley et al., 2015

HI (right CCA ligation and FiO2 of 8%)

32
C rectal temperature in cooling chamber

Chen et al., 20156

Temporary MCAO (120 minutes) by microcatheter

30
C brain temperature after SD rats MCAO by cold saline infusion via microcatheter

Yuan et al., 20137

CPB, hypothermic circulatory Deep hypothermia arrest

Zgavc et al., 20138

MCAO (endothelin-1 injection)

5

Newborn rats (8e13 days)

CBF During/After Hypothermia CBF (spectroscopy) reduced by >20% during hypothermia

Neuroprotection Significantly lower brain damage at 1 and 4 weeks after HI

Reduction of early postischemic Improved neurologic function hyperperfusion, improvement of and decreased infarct volume postischemic hypoperfusion after 48 hours

Rats

Significant reduction of CBF (laser-speckle-imaging) during cooling

33
C brain temperature, surface cooling

Wistar rats (270e300 g)

Nonsignificant reduction of CBF Significant reduction of infarct (LDF) by hypothermia volume

Li et al., 20119

None (cooling procedure, 32
C rectal temperature, reactivity of CBF to hindpaw surface cooling stimulation)

SD rats (270  20 g)

Response of CBF (laser-speckle imaging) at 32
C decreased and delayed; smaller area of response

Cheng et al., 201110

HI (bilateral CCA ligation and FiO2 of 6%)

32
C nasopharyngeal temperature, selective head cooling

Newborn pigs (7 days)

CBF (microspheres) significantly Decrease of brain energy reduced at 35
C and 32
C consumption, improvement of versus normothermic controls AVDO2, glucose, and lactate

32
C brain temperature, surface cooling

SD rats (320 g) CBF (LDF) improved during Decrease of glucose posthemorrhage hypoperfusion; depletion, lactate autoregulation preserved accumulation, and glutamate release after SAH

Schubert et al., 200811 SAH by cisterna magna blood injection

Royl et al., 200812

None (CBF/CMRO2 coupling 27
C brain temperature, under hypothermia) surface cooling

Ouchi et al., 200613

None (CBF/CMRO2 under propofol and hypothermia)

Bedell et al., 200414

TBI (fluid percussion injury) 32
C brain temperature, surface cooling

SD rats (450e650 g)

Reduction of CBF (LDF) during hypothermia without and with FPI

33
C brain temperature, surface cooling for 120 minutes starting before ischemia

SD rats (250e300 g)

CBF (LDF) reduced by 20% during Improved neurologic cooling; increase by rewarming performance and reduced contralateral but not ipsilateral infarct volume to MCAO

Sonn et al., 200216

None (reaction of CBF and 32
C brain temperature, cortical function in awake external cooling and anesthetized animals)

Wistar rats (180e250 g)

No significant change of CBF (brain function multiprobe) in awake and anesthetized rats

Ehrlich et al., 200217

CPB

28
C, 18
C, and 8
C brain temperature on CPB

Pigs (7e13 kg) CBF (microspheres) continuously CMRO2 at 28
C 50% of reduced from 37
C down to baseline 18
C

Okubo et al., 200118

None (CBF/CMRO2 during cooling procedure)

32
C brain temperature, surface cooling

Newborn pigs (1.5e2 kg)

Laptook et al, 200119

None(effects of cooling on Approximately 34
C brain Newborn pigs CBF by total body cooling temperature, surface cooling (1.4  0.4 kg) and selective head cooling) of head and body

Schöller et al., 200415 Permanent MCAO

35
C rectal temperature, surface cooling

Wistar rats (250e300 g)

CBF (LDF) decreased by approximately 50%

Rhesus monkeys Reduction of CBF (positron emission tomography) during cooling procedure

Wistar rats Jenkins et al., 200120 HI (bilateral CCA clamping) 34
C rectal temperature, and hemorrhagic surface cooling induced before (270e370 g) hypotension ischemia

Decrease of CMRO2 higher than decrease of CBF; metabolic coupling preserved Constant ratio of CBF/CMRO2

CBF reduced to 41% of baseline Reduction of CMRO2 and at 32
C CMRglu by 53% and 46% CBF (microspheres) reduced by 40% by both selective head cooling and total body cooling CBF (laser-Doppler flowmetry) improved during and after ischemia

Faster recovery of EEG activity and less hippocampal damage in hypothermic animals Continues

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Table 1. Continued Experimental Model of Brain Ischemia/Trauma

Reference 21

Temperature


Species




Walter et al., 2000

None (selective brain cooling procedure with sequential rewarming)

30 C and 25 C (bilateral CCA Juvenile pigs perfusion after extracorporeal blood cooling)

Krafft et al., 200022

None (CBF and glucose 35
C and 32
C brain utilization under temperature hypothermia and isoflurane)

Westermaier et al., 200023

Temporary (90 minutes) MCAO

Mori et al., 199924

CBF During/After Hypothermia

Neuroprotection

CBF decreased by 44% and 73% Reduction of CMRO2 by at 30
C and 25
C hypothermia with decrease of Q10 ratio at 25
C

SD rats (318  18 g)

CBF (autoradiography) increased by isoflurane to 129%, reduced to baseline at 35
C, and reduced to 70% of baseline at 32
C

33
C brain temperature, surface cooling

SD rats (250e300 g)

CBF (LDF) reduced by cooling; increase to baseline by rewarming contralateral to MCAO; attenuation of postischemic hyperperfusion

Significant reduction of infarct volume in hypothermic animals

None (CBV, CMRO2, CVR under hypothermia)

25
C brain temperature, surface cooling

Cats

CBF (hydrogen clearance) reduced by 88% at 25
C

Decrease of CMRO2, increase of AVDO2. Increased CBF by noradrenalin

Burger et al., 199825

Epidural mass lesion (balloon inflation, 30 minutes)

32
C body temperature, surface cooling

SD rats

CBF (laser-Doppler flowmetry) Lower ICP and faster recovery improved during balloon inflation of SEP in hypothermic animals and after deflation

Mori et al., 199826

Epidural mass lesion 33
C and 29
C after release of Cats (balloon inflation, 5 minutes balloon inflation, surface to CPP 0) cooling

Niwa et al., 199827

None (CBF/autoregulation during hypothermia)

33
C brain and rectal Wistar rats temperature, surface cooling (350e400 g)

Klementavicious et al., None (CBF and metabolic 28
C, surface cooling parameters during cooling) 199628

Wistar rats (350e550 g)

CBF (hydrogen clearance) during postinflation hyperemia reduced by 56% at 33
C and 77% at 29
C

CMRO2 and CMRglu significantly decreased; bloodbrain barrier breakdown attenuated

CBF (autoradiography) lower under hypothermia

Cerebrovascular autoregulation disturbed

CBF (hydrogen clearance) continuously reduced by cooling from 38
C to 28
C

Hoffman et al., 199629 HI (right CCA ligation and hemorrhagic hypotension)

34
C and 31
C rectal SD rats temperature, surface cooling (350e450 g)

CBF (LDF) unchanged by hypothermia before ischemia

Jiang et al., 199430

Transient (120 minutes) MCAO

30
C body temperature, surface cooling

Wistar rats (270e310 g)

CBF (1H-magnetic resonance imaging perfusion maps) reduced before and after ischemia

Kuluz et al., 199331

None (CBF under selective brain cooling)

31
C brain temperature by surface cooling of the head

Wistar rats 270e360 g

CBF (LDF) increased by 100% during cooling period, decrease by rewarming

30
C brain temperature, surface cooling

SD rats (300e480 g)

CBF (LDF) decreased during and Reduced cortical damage in after ischemia in hypothermic transient and permanent animals MCAO

Morikawa et al., 199232 Transient (120 minutes) MCAO and permanent MCAO

Baldwin et al., 199133 Global ischemia (20 30
C brain temperature after Beagle dogs minutes), rapid increase of ischemia by reinfusion of (10e13 kg) ICP by artificial cooled blood cerebrospinal fluid infusion and withdrawal of blood Palmer et al., 198934

None (CBF and CMRO2 during cooling)

20
C rectal temperature, surface cooling

Better EEG recovery and less tissue damage in hypothermic animals

CBF (microspheres) improved Significantly better recovery of after 30 minutes of reperfusion SEP amplitude and latency in hypothermic animals; no after hypothermic reperfusion significant difference after 60, 90, and 120 minutes

Newborn dogs CBF (autoradiography) significantly lower in all brain regions

CMRglu significantly reduced during hypothermia

HI, hypoxia-ischemia; CCA, common carotid artery; FIO2, inspiratory fraction of oxygen; CBF, cerebral blood flow; MCAO, middle cerebral artery occlusion; SD rats, Sprague-Dawley rats; CPB, cardiopulmonary bypass; LDF, laser-Doppler flowmetry; AVDO2, difference of arterial and cerebral venous oxygen content; CMRO2, cerebral metabolic rate for oxygen; TBI, traumatic brain injury; CMRglu, cerebral metabolic rate for glucose; EEG, electroencephalogram; ICP, intracranial pressure; SEP, somatosensory-evoked potential; CPP, cerebral perfusion pressure; CSF, cerebrospinal fluid. Continues

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Table 1. Continued Experimental Model of Brain Ischemia/Trauma

Reference 3

Temperature


34 C rectal temperature, surface cooling

Species

Busija et al., 1987

None (CBF and CMRO2 during cooling)

Newborn pigs (1e2 kg)

Török et al., 201135

SAH (endovascular filament 33
C brain temperature 1e3 SD rats model) hours and 3e5 hours after (250e300 g) SAH

CBF During/After Hypothermia

Neuroprotection

CBF (microspheres) reduced CMRO2 significantly reduced, (40%e50%) in all areas of the exceeding the reduction of brain CBF CBF (laser-Doppler flowmetry) not significantly altered by hypothermia

Significantly better neurologic performance after 24 hours

HI, hypoxia-ischemia; CCA, common carotid artery; FIO2, inspiratory fraction of oxygen; CBF, cerebral blood flow; MCAO, middle cerebral artery occlusion; SD rats, Sprague-Dawley rats; CPB, cardiopulmonary bypass; LDF, laser-Doppler flowmetry; AVDO2, difference of arterial and cerebral venous oxygen content; CMRO2, cerebral metabolic rate for oxygen; TBI, traumatic brain injury; CMRglu, cerebral metabolic rate for glucose; EEG, electroencephalogram; ICP, intracranial pressure; SEP, somatosensory-evoked potential; CPP, cerebral perfusion pressure; CSF, cerebrospinal fluid.

patients are admitted to hospital, a diagnosis is found, and treatment can be started. Experimental studies on the neuroprotective effect of mild hypothermia after subarachnoid hemorrhage (SAH) are rare (because SAH differs from ischemic stroke and traumatic brain injury in its pathophysiology) and therapeutic mild hypothermia may simply have been initiated too late. Different from ischemic stroke and traumatic brain injury, where ischemia and primary brain injury usually occur simultaneously, ischemic lesions in patients with aneurysmal SAH and post-SAH vasospasm usually occur as delayed cerebral ischemia, because the initial global ischemia is followed by a long-lasting and persistent low-flow status, which bears the danger of progressive energy exhaustion. Therefore, timing is definitely the issue in our perspective. Applied early enough, mild hypothermia may prevent energy depletion and have a neuroprotective effect. In a small clinical series, Karamatsu et al.38 found that patients with SAH may profit from treatment with very mild hypothermia if started early after SAH. The new idea in our hypothesis is that mild hypothermia, applied early enough within the right time frame in the early hours after SAH in which the low-flow status is still happening and delayed ischemia is still ahead of the patient, could therefore have a neuroprotective effect. The present study was conducted in the framework of a larger-scale project assessing the neuroprotective properties of various treatment forms in the early stage of SAH. It investigated the effects of mild hypothermia induced in the first minutes after experimental SAH in rats on hemodynamic parameters, functional outcome, and tissue damage. METHODS For the experiments, 20 male Sprague-Dawley rats (300e350 g body weight), purchased from Charles-River, Sulzfeld, Germany were used. All experiments were approved by the regional authorities and the district government of Bavaria, Germany. Animal Preparation and Monitoring The animals were anesthetized with 4% isoflurane, orally intubated and mechanically ventilated (pressure-controlled mode) with an air/oxygen mixture to maintain normal arterial blood

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gases. After induction of anesthesia, isoflurane was reduced to 3% for surgical procedures and to 2% from 30 minutes before SAH until the end of the monitoring period. A temporalis muscle probe was used to monitor brain temperature throughout the experiment. A thermostatically regulated, feedback-controlled heating lamp was used to maintain the temporalis muscle and rectal temperature at 37.0
C. The tail artery was cannulated for continuous measurement of mean arterial blood pressure (MABP) and for blood sampling. Arterial blood gases were measured 30 minutes and 5 minutes before and in hourly intervals after induction of SAH. Laser-Doppler Flowmetry and Intracranial Pressure A 2-channel laser-Doppler flowmeter (LDF) (MBF3D [Moor Instruments, Axminster, United Kingdom]) was used for continuous bilateral monitoring of local cerebral blood flow (LCBF) in the area of the cerebral cortex supplied by the middle cerebral artery (MCA). To place the LDF probes, burr holes were drilled 5 mm lateral and 2 mm posterior to the bregma without injury to the dura mater. For measurement of intracranial pressure (ICP), a third burr hole was drilled over the left frontal cortex 3 mm lateral and 0.5 mm anterior to the bregma. After all burr holes were completed, the animals were placed in a supine position with their head fixed in a stereotactic frame with nonperforating ear bars. Rectangular bent laser-Doppler probes were positioned in the posterior burr holes with a micromanipulator. An intraparenchymal Camino ICP probe (Integra Neurosciences, Plainsboro, New Jersey, USA) was advanced 2 mm into the brain by the use of a third micromanipulator. Induction of SAH SAH was induced by the endovascular puncture method.39,40 After surgical exposure of the right cervical carotid bifurcation, temporary aneurysm clips were placed on the common and internal carotid artery (ICA). A 3-0 Prolene filament (Ethicon, Inc., Somerville, New Jersey, USA) was inserted into the external carotid artery and fixed with a silk ligature and the temporary clips were removed. After a stabilization period of 30 minutes, the filament was advanced into the ICA until ipsilateral LCBF decreased, indicating that the tip of the filament was at the intracranial

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division of the ICA occluding the origin of the MCA. The filament then was pushed 2e3 mm further for intracranial vessel perforation in the ICA/anterior communicating artery region. The suture was quickly withdrawn into the external carotid artery to ascertain reperfusion and development of SAH. SAH was indicated by a rapid bilateral decrease of LCBF and increase of ICP. Experimental Groups The rats were randomly assigned to 1 of 2 groups (n ¼ 10 for each group): 1) normothermia: the animals’ rectal and temporalis muscle temperature was maintained at 37.0
C from 30 minutes before induction of SAH until the end of the monitoring period. 2) hypothermia: the animals’ body temperature was reduced from 37.0
C to 34
C within 15 minutes by external cooling using icepacks positioned on both sides of the trunk. The feedbackcontrolled heating lamp was downregulated to maintain 34
C until 180 minutes after SAH. Thereafter, the animals were rewarmed to 37.0
C again by upregulation of the feedbackcontrolled heating system. Termination of the Experiment and Wound Closure At the end of the monitoring period, the ICP probe, laser-Doppler probes, and the arterial line were removed and the wounds were closed with a skin suture. Isoflurane was withdrawn and the animals were allowed to wake up and were placed back in their home cage. Neurologic Assessment Twenty-four hours later, these animals underwent neurologic testing, including the assessment of hemiparesis and activity. For neurologic examination, the animal was placed into a large uncovered cage. After 10 minutes of acclimatization, the presence of a hemiparesis was assessed using a 6-grade modification of the scoring system introduced by Bederson et al.41,42 Activity was evaluated after repeated manipulation (tail-holding, lateral push, repeated displacement of the animal) and graded following a 5-grade scale: 4, normal spontaneous activity; 3, slightly reduced spontaneous activity; 2, little or no spontaneous activity, but reaction on stimulus; 1, no activity on stimulus; and 0, animal dead.43 Neurologic assessment was performed by an examiner blinded to the animal’s treatment arm. Histologic Assessment After neurologic assessment, the animals were again anesthetized with isoflurane followed by an intraperitoneal injection of 50 mg sodium-thiopental. The animals were then transcardially perfused with 4% paraformaldehyde, the brains removed, and the amount of subarachnoid blood was determined using a semiquantitative scale as follows: 0, no blood visible; 1, traces of blood visible, no blood clot: 2, unilateral clot; 3, generalized bilateral basal blood clot; and 4, intracerebral hematoma with or without subarachnoid blood. Thereafter, the brains were embedded in paraffin and cut into 4-mm-thick coronal sections at 400-mm intervals, and the brain slices were stained with hematoxylin-eosin (HE). Three defined parts of the CA1 region of the hippocampus (bregma 3.24, 4.92, 6.12) were determined according to a

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stereotactic atlas of the brain,44 bilaterally analyzed for vital and injured neurons, and their number per field (0.2  0.3 mm) was counted by an investigator blinded toward the group allocation. Vital neurons were defined as cells with basophilic cytoplasm and intact nuclei, whereas cells were counted as injured neurons if they were composed of eosinophilic cytoplasm and presented with pyknotic nuclei, karyorrhexis, or karyolysis.45,46 For immunostaining, brain sections were washed in phosphatebuffered saline (PBS), blocked in a solution of 10% normal horse serum in PBS. Thereafter, the primary antibody (Cleaved Caspase3 antibody Asp175, 1:300 [Cell Signalling Technology, Cambridge, United Kingdom]) was added and the sections were incubated at 4
C overnight. Sections were then again washed with PBS, the secondary antibody (Cy3 conjugate goat and rabbit IgG [Jackson Immunoresearch Laboratories Inc., Suffolk, United Kingdom]) was added and again incubated for 1 hour. After, the sections were again washed with PBS. The sections were costained with 40 ,6-diamidin-2-phenylindol and scanned for caspase-3epositive cells per visual field under 40-fold magnification using a fluorescence microscope (Leica DMI 3000B [Leica Microsystems, Wetzlar, Germany]). The number of damaged cells is presented as a percentage of all visible cells in the field of view (Figure 1). Statistical Analysis Statistical analysis was performed with GraphPad Prism 4 (GraphPad Software, Inc., La Jolla, California, USA). Physiologic data for each time point, LDF, ICP, and PtiO2 data were analyzed by an unpaired t test. When indicated, data were tested for normal distribution using a D’Agostino and Pearson Normality Test. A P value of < 0.05 was considered significant. Results are presented as mean  standard deviation. RESULTS Physiologic Parameters Values of pH, PaCO2 (arterial tension of carbon dioxide), and PaO2 (arterial tension of oxygen) are presented in Table 2. Differences between the groups were not significant. MABP, ICP, Cerebral Perfusion Pressure MABP increased in the control group after the induction of SAH from a baseline of 83  19 mm Hg to a maximum of 89  15 mm Hg after 5 minutes and then declined to 78  17 mm Hg and 78  22 after 1 and 3 hours. In the hypothermia group, MABP increased from a baseline of 85  23 mm Hg to a maximum of 87  22 mm Hg 30 minutes after SAH and then decreased to 74  19 mm Hg and 66  16 mm Hg after 1 and 3 hours, respectively. The difference of MABP between the 2 groups was not statistically significant at any time point after induction of SAH (Figure 2A). In the control group, ICP increased from a baseline of 9  6 mm Hg to a maximum of 46  17 mm Hg 1 minute after SAH, declined to 20  9 mm Hg after 1 hour and to 21  9 mm Hg at the end of the observation period. In the hypothermia group, baseline ICP was 8  2 mm Hg before induction of SAH and increased to 42  24 mm Hg 1 minute after SAH, then declined to 8  6 mm Hg after 60 minutes and 11  6 at the end of the observation period. The difference between the 2 groups was significant from 15

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Figure 1. (AeD) Examples of histologic and immunohistochemical changes in the hippocampal CA1 field. (A) White arrows indicate cells with pycnotic nucleus and karyolysis. (B) 40 ,6-Diamidin-2-phenylindol

minutes after SAH until the end of the monitoring period (Figure 2B). In the control group, cerebral perfusion pressure (CPP) decreased from a baseline of 74  23 mm Hg to a minimum of 28

Table 2. Arterial Blood Gases Before Induction of Subarachnoid Hemorrhage and in Hourly Intervals Thereafter Arterial Blood Gases

pH

PaCO2 (mm Hg)

PaO2 (mm Hg)

Normothermia Before SAH

7.39  0.03

40.8  5.0

107.8  15.3

60 minutes after SAH

7.40  0.03

39.1  5.3

111.6  23.0

120 minutes after SAH

7.41  0,04

37.5  5.5

114.1  16.1

7.39  0.04

38.2  5.7

111.4  10.2

Hypothermia Before SAH 60 minutes after SAH

7.38  0.04

36.7  7.8

132.1  23.3

120 minutes after SAH

7.37  0.04

40.6  6.5

116.9  27.7

If blood gases were not within the normal range after the end of surgical procedures, mechanical respiration was adjusted and measured again 15 minutes later. SAH was induced if blood gases were in the normal range. SAH, subarachnoid hemorrhage.

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(DAPI) staining, (C) caspase-3 staining, and (D) overlay of DAPI and caspase-3 immunostaining with white arrows showing caspase-3 positive CA1-cells indicated by costaining of DAPI and caspase-3.

 21 mm Hg after 1 minute and then recovered to 58  24 and 57  23 mm Hg after 1 and 3 hours. In the hypothermia group, CPP decreased from a baseline of 77  30 mm Hg to 35  25 mm Hg and then recovered to 66  20 mm Hg and 55  19 mm Hg after 1 and 3 hours, respectively. The difference between the groups was not significant at any time point after SAH (Figure 2C). LCBF The ipsilateral LCBF (right hemisphere) decreased to a minimum of 25.8%  18.6% of baseline 1 minute after SAH and recovered to 55.7%  42.3% and 87.7%  47.6% of baseline 1 and 3 hours after SAH in the control group. In the hypothermia group, the ipsilateral LCBF decreased to a minimum of 21.8  18.5% of baseline 1 minute after SAH and recovered to 68.8  64.8% and 85.7  45.6% of baseline 1 and 3 hours after SAH. The differences were not statistically significant (Figure 3A). In the control group, the contralateral LCBF (left hemisphere) declined to a minimum of 32.1%  22.5% of baseline 1 minute after SAH and recovered to 56.6%  14.0% after 60 minutes and increased further to 79.2%  22.8% at the end of the observation period. In the hypothermia group, the contralateral LCBF decreased to 34.6%  19.1% of baseline 1 minute after SAH and returned to 46.2%  27.0% and to 66.4%  39.9% of baseline 1 and 3 hours after SAH, respectively. The differences were significant from 120 to 180 minutes after SAH (Figure 3B).

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Figure 3. (A and B) Courses of local cerebral blood flow (LCBF) over the right (ipsilateral) and left (contralateral) hemisphere continuously measured by laser-Doppler flowmetry from 30 minutes before until 180 minutes after right-sided intracranial vessel perforation by the endovascular filament model. Due to a lower ICP and higher CPP, we observed a trend towards higher LCBF early after vessel perforation in the hypothermia-group. Later in the monitoring phase, there was a tendency of lower LCBF in hypothermic animals, presumably due to a metabolic coupling.

Figure 2. Courses of (A) mean arterial blood pressure (MABP), (B) intracranial pressure (ICP), and (C) cerebral perfusion pressure (CPP), continuously monitored from 30 minutes before until 180 minutes after induction of subarachnoid hemorrhage (SAH) by the endovascular filament model. Intracranial pressure was significantly lower in hypothermic animals. Differences in cerebral perfusion pressure were not significantly different (*P < 0.05).

Mortality and Neurologic Performance One animal assigned to the control group died during the induction of anesthesia. One animal in the treatment group required several intubation attempts. After an uneventful course of the experiment and extubation, the animal developed severe respiratory problems and had to be killed shortly after withdrawal of anesthesia despite a good initial neurologic performance. Laryngoscopy showed massive laryngeal swelling and pharyngeal hemorrhage, caused by repeated intubation attempts. No further animal had to be excluded from the study or died within the designated survival time of 24 hours after induction of SAH. Although all animals recovered well from anesthesia and were able

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to eat food pellets and drink water appropriately, all animals in both groups lost weight in the first 24 hours after SAH. The animals in the hypothermia group lost 10.9  2.7% of their body weight and the animals in the control group lost 12.4  3.3% of their body weight. In the hypothermia group, all animals were judged to have normal activity by a blinded investigator (median, 4.0). In the control group, 6 animals had slightly reduced activity, 2 animals showed activity only after external stimuli, and 1 animal showed no activity (median, 3.0). The difference was not statistically significant (P < 0.01, Figure 4). Extent of Hemorrhage and Histologic Damage The extent of subarachnoid blood did not significantly differ between the 2 groups. According to the semiquantitative score, 4 animals in the control group were classified as grade 1, 3 animals as grade 2, and 3 animals as grade 3. In the hypothermia group, 1 animal was classified as grade 1, 5 animals as grade 2, and 4 animals as grade 3. In the control group, 13.7%  6.8% of neurons in the hippocampal CA1 field were counted as injured neurons by histologic assessment (HE staining), compared with 4.6%  3.1% in the hypothermia-treated group (P < 0.01, Figure 5). No signs of territorial or lacunar infarctions were visible in either hemisphere as a sign of vessel occlusion or cerebral vasospasm. In the control

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Figure 6. Caspase-3epositive CA1 cells. The number of caspase-3epositive cells, depicted as ratio of total cell count per visual field, was not significantly reduced by treatment with mild hypothermia. Figure 4. Activity score. Neurologic performance assessed 24 hours after induction of subarachnoid hemorrhage (SAH) by a 5-scale activity score. All animals treated by mild hypothermia showed a normal neurologic examination result. (*P < 0.05).

group, 7.9%  2.0% of all counted neurons were caspase-3 positive, compared with 7.0  2.9% in the hypothermia-treated group (P < 0.05, Figure 6).

DISCUSSION Like many other experimental studies of various kinds of brain damage, the results of this study show a strong neuroprotective effect of moderate hypothermia in an animal model of SAH. The experiments were conducted in the framework of a project investigating various forms of emergency treatment in the acute phase of SAH that can potentially be transferred into clinical emergency medicine. For this purpose, well-established drugs and treatment measures are evaluated for their ability to attenuate neurologic and histologic damage after SAH. To qualify for acute treatment, this form of therapy must be readily available, easy to

Figure 5. Hippocampal damage. Histologic assessment (hematoxylin-eosin staining) showed a significant reduction of cell damage in the hippocampal CA1 field. Values represent the rate of damaged cells of the total cell count per visual field under 40-fold magnification (**P < 0.01).

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perform, and not be contraindicated in any possible differential diagnosis. The latter include embolic and hemorrhagic stroke, hidden traumatic brain injury, intoxication, seizures, heart attack, or metabolic reasons for a loss of consciousness. Mild hypothermia fulfills these criteria because its risk profile is well known and it is not contraindicated in any of the reasons for a loss of consciousness.47 It is feasible and could be used as a first-tier therapy in patients immediately after SAH. In the present study, we found a trend to higher blood pressure values during the cooling period and significantly lower ICP values in hypothermic animals, resulting in a higher CPP in the first hour after SAH. A positive inotropic effect mediated by an improvement of myocardial contractility has recently been described during the cooling period in an experimental model.48 In that study, the investigators suggested that cooling could reduce the need for inotropic drugs. Apart from primary neuroprotection, this effect may be welcome, particularly in patients with severe SAH who may develop cardiac instability and hypotension with or without subendocardial ischemia.49,50 The ICP-lowering effect of mild and moderate hypothermia has been extensively studied in recent years51,52 and is, by itself, a beneficial effect in the face of increased ICP after SAH. Aneurysm rupture has only rarely been observed under monitoring conditions. However, the course of physiologic parameters in the acute phase of SAH has been characterized in experimental models by several groups.11,23,35,39,40 It has consistently been observed that ICP quickly declines after an initial peak to remain moderately increased for several hours after induction of SAH. In consequence, CPP recovers well after an initial steep decline. Thus, the first hour after SAH is likely to be the most effective time to apply therapeutic hypothermia. Because ischemic brain damage is the function of duration and severity of ischemia,53 the induction of hypothermia in the first minutes after SAH would be the earliest possible and ideal time point of treatment. Depending on the clinical situation and circumstances, active cooling may be necessary or not. The induction of general anesthesia for intubation in poor-grade patients with SAH in the field may, by itself, favor cooling of the patient and active cooling may not even be necessary.54 Török et al.35 investigated the effects of early (1e3 hours after SAH) and late (3e5 hours after SAH) hypothermia, induced by

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vessel perforation. These investigators found a strong neuroprotective effect of early moderate hypothermia (33
C). Although still beneficial, later induction of hypothermia offered less neuroprotection, suggesting that the earlier cooling is performed, the more beneficial it is for neuroprotection purposes. Schubert et al.11 investigated the effects of moderate hypothermia on physiologic parameters, CBF, and tissue metabolites in a cisterna magna injection model of SAH. Using cerebral microdialysis, these investigators found an improvement of metabolic exhaustion and release of excitatory amino acids by moderate hypothermia of 32
C. However, whether metabolic improvement resulted in reduced tissue damage was not quantified in that study. Compared with normothermic controls, the acute hypoperfusion after blood injection was ameliorated in hypothermic animals.11 This differs from our findings, which only showed a marked difference between the normothermic control group and the treatment group in the immediate posthemorrhagic period, which is within the cooling period. Thereafter, it tended to be lower in hypothermic animals after the target temperature of 34
C was reached. Also Choi et al. showed recently in a small prospective pilot study that hypothermia reduced vasospasm and delayed cerebral ischemia in the hypothermia group, as well as an improvement of functional outcome and reduction of mortality in the hypothermia group.55 Still, they started hypothermia after successful coiling or clipping and may therefore just miss the optimal time frame for neuroprotection. In our hypothesis, we were thinking of investigating the possible neuroprotective effect of hypothermia in the first 3 hours after SAH, where ICP and CPP are almost back to normal levels, cerebral blood flow (CBF) is still reduced down to 60% and lasts for several hours, and delayed ischemia is still ahead of the patient. Our data of increase in MAP and CPP with consecutive increase of CBF and attenuation of neuronal injury support our hypothesis that mild hypothermia could have a neuroprotective effect if applied early enough within the right time frame in the early hours after SAH. Although experimental studies investigating the effects of whole-body hypothermia without a specific kind of brain lesion unequivocally found a reduction of CBF as a result of metabolic coupling,12,13,18 the effects of hypothermia on CBF after ischemic or traumatic brain lesions or SAH show a wider spectrum of findings. Although some investigators report a reduction5,8,10,24,30,32 or no change29 of CBF by hypothermia in experimental models of global and focal brain ischemia and traumatic brain injury, others report an improvement of CBF during and after ischemia20 or trauma, respectively.25 In experimental studies of transient MCA occlusion, the immediate postischemic hyperperfusion, which is likely to contribute to ischemic damage (e.g., by hemorrhagic transformation of ischemic territories and free radical induced injury) and the delayed perfusion deficit preventing a cleaning of metabolic products and correction of the metabolic state, have been reported to be reduced by treatment with mild hypothermia6,23 (Table 2). These findings may be sign of a preserved autoregulation after ischemic events.

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Baldwin et al.33 observed an attenuation of early postischemic hypoperfusion (30 minutes after reperfusion) in a dog model of global ischemia by moderate hypothermia in which there was no marked influence regarding the immediate hyperperfusion or later time points of delayed post-ischemic hypoperfusion. Although the pathophysiology of global ischemia with reperfusion differs from that of SAH concerning the extent and duration of ischemia and the timing, course, and completeness of reperfusion, certain parallels can be observed. Baldwin et al. did not use a model of vessel occlusion to produce ischemia but intraventricular infusion of artificial CSF until CPP came to zero. Reperfusion, in turn, was the result of a reduction of CSF and recovery of CPP. The effect of hypothermia on CSF might strongly depend on the experimental model and setup used. The fact that Schubert et al.,11 in contrast to our own results, found a sustained increase of CBF after SAH may be the result of a different model of SAH produced by injection of blood into the subarachnoid space, which is highly standardized and temporarily decreases CBF to zero but has been shown to result in a less profound overall recovery of CBF than has the endovascular perforation model.56 This may also result in distinct differences regarding the effect of neuroprotective and CBF-modulating measures and drugs. The neuroprotective effect of mild hypothermia in an experimental model of brain injury has once again been shown by the present study. However, we found a discrepancy between cell damage visible in HE-stained brain slices and caspase-3 immunostaining. Ordy et al.57 analyzed the progression of cell damage in the CA1 field of the hippocampus observing a detectable CA1 pyramidal cell loss even at day 1 after 4-vessel occlusion in the rat. Cell damage distinctly increased in the following days. Histologic cell damage in the control group of this study was in the range of that of previous studies of our group.58 Cell damage after 24 hours was strongly reduced in hypothermic animals. However, immunostaining showed only a marginal difference in caspase-3epositive cells as a marker of apoptosis, indicating that temporary hypothermia only in the first hours after SAH is not able to prevent the growth of pyramidal cell damage. The 3-hour period of hypothermia may have been too short in this study to have a protective effect on the secondary growth of selective hippocampal damage. Temporary postischemic or posthemorrhage hypothermia may only postpone some of the secondary lesion growth. CONCLUSIONS This study shows that the induction of mild hypothermia immediately after experimental SAH can reduce ICP and improve CBF in the early course of the disease. Although hypothermia may not inhibit lesion growth, it has shown its neuroprotective effect in this study. Because there is no relevant contraindication for mild hypothermia in any thinkable differential diagnosis to SAH, it may readily be applied in patients as a first-tier therapy and could even be used by emergency rescue services before the patient is admitted to hospital. In addition, studies of cerebral ischemia have shown that the effect of hypothermia may be enhanced by combination with other substances or methods exerting additive effects.59

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Conflict of interest statement: T.W. received lecture fees from Medtronic. All other authors have no conflict to declare. Received 14 March 2017; accepted 16 September 2017 Citation: World Neurosurg. (2017). https://doi.org/10.1016/j.wneu.2017.09.109 Journal homepage: www.WORLDNEUROSURGERY.org Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2017 Elsevier Inc. All rights reserved.

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