Brain Poster Session: Neuroprotection—Hypothermia

314. Brain cooling by novel intraventricular catheter

R. Moomiaie-Qajar¹, G. Gould¹, D. Botta², J. Simmons³ and J. Elefteriades⁴

¹Yale School of Medicine; ²Surgery, Yale School of Medicine, New Haven; ³CoolSpine LLC, Woodbury; ⁴Surgery-Cardiac, Yale School of Medicine, New Haven, Connecticut, USA

Background: The neuroprotective effects of hypothermia are well-known. We recently reported effective cooling of the spinal cord in a sheep model by a self-contained intrathecal catheter. In that study we sucessfully induced localized hypothermia of the spinal cord, thereby promising a potential modality for spinal cord protection during aortic aneurysn repair.

Purpose: The present study is designed to determine if cooling catheters in the lateral ventricles of the brain can effectively cool the cerebral spinal fluid (CSF) and thereby reduce brain temperature while maintaining systemic normothermia. In particular, it is unknown whether a cooling system can overcome the warming by the native cerebral blood flow.

Methods: The cooling catheter is a self-contained system that circulates a cold fluid and cools the CSF that circulates in the brain. The CSF in turn cools the surrounding brain by conduction. This cooling catheter was specifically designed for application to the lateral ventricles of the brain. Burr holes were made in the skull and the catheter was placed into the lateral ventricles using the standard method for placement of ventriculostomy catheter. The study was conducted in sheep because of their body mass is similar to adult humans. To monitor the cooling effect, four temperature probes were placed in the brain (left and right hemispheres of the brain in anterior and posterior locations to the ventricles).

Results: Five experiments were successfully completed (temperature probes modified after first experiment). In each animal, two cooling catheters were successfully placed into the lateral ventricles. The mean brain temperature for all sheep decreased to 34.5°C (mean) during the 3 h cooling period. This represented a 9.7% reduction from the average baseline brain temperature of 38.2°C. During the cooling period, the cooling fluid was circulated through the catheter at a maximum rate of 50 mL per minute. The lowest achieved brain temperature during cooling was 26.7°C, which represented a 28.6% decrease from baseline. When cooling was stopped, the brain temperature readings equilibrated with the core temperature promptly. Post-mortem examination of the brains showed no significant morphologic changes under gross or histologic examinations.

Conclusion: Localized cooling of the brain to moderate hypothermic levels while maintaining relative systemic normothermia was demonstrated in an animal model with our intraventricular cooling catheters. This novel technique holds promise as an additional neuroprotection modality to mitigate brain injury in deep hypothermic circulatory arrest for aortic arch surgery as well as in traumatic brain injury and stroke.

Figure

CoolSpine intraventricular cooling catheter.

552. The effect of FNK protein and hypothermia combined therapy on rat focal brain ischemia model

M. Sakurazawa¹, K. Katsura¹, M. Saito¹, S. Asoh², S. Ohta² and Y. Katayama¹

¹Division of Neurology, Nephrology and Rheumatology, Department of Internal Medicine, Nippon Medical School, Tokyo; ²Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Nippon Medical School, Kawasaki, Japan

Objective: We previously reported that PTD-FNK protein has a strong neuroprotective effect on rat focal brain ischemia models. ¹ FNK protein is derived from antiapoptotic protein Bcl-x_L by substituting three amino acids artificially and thereby gains a higher anti cell death activity. FNK protein was fused with PTD (protein transduction domain) of the HIV/Tat protein to be able to pass through cell membranes and it was shown to be transduced to neuronal cells rapidly. The aim of this study is to investigate the effect of PTD-FNK protein and hypothermia combined therapy on cerebral infarction.

Methods: Eight-week-old SD rats were subjected to a 90 mins middle cerebral artery occlusion (MCAO). Focal ischemia was produced by intraluminal occlusion of the left MCA with a nylon monofilament. Rats were divided into 4 groups:

• (1)

  37°C Vehicle administration,

• (2)

  37°C PTD-FNK administration,

• (3)

  35°C Vehicle administration, and

• (4)

  35°C PTD-FNK administration.

PTD-FNK protein was intravenously administered 60 mins after the induction of MCAO. Hypothermia (35°C) was applied during 90 mins MCAO. At 24 h after MCA occlusion, the brain was removed and sliced into six coronal sections. All six sections were stained with 3% of 2,3,5-triphenyltetrazolium chloride and the volume of infarction was measured.

Results: Infarct volumes were significantly reduced in groups of (2) (142.5±49.1 mm³), (3) (81.5±35.0 mm³) and (4) (77.2±60.9 mm³) against group (1) (190.6±50.4 mm³). We failed to find significant difference of infarct volumes between group (3) and (4).

Conclusions: In this study we failed to show an additional protective effect with PTD-FNK protein administration compared to hypothermia alone therapy. If the hypothermia therapy is enough protective as shown in this study, it may be hard to get additional protective effect with additional therapy. Besides, possibility of reduced transduction of PTD-FNK into neuronal cells during hypothermia might influence the results.

695. When hypothermia meets hypotension and hyperglycemia: the diverse effects of adenosine 5′-monophosphate on cerebral ischemia in rats

F. Zhang^1,2, S. Wang¹, Y. Luo^3,4, X. Ji^3,4, E.M. Memoto⁵ and J. Chen^1,2

¹Neurology and Center of Cerebrovascular Disease Research, University of Pittsburgh; ²Geriatric Research, Educational and Clinical Center, Veterans Affairs Pittsburgh Health Care System, Pittsburgh, Pennsylvania, USA; ³Cerebrovascular Diseases Research Institute, Xuanwu Hospital of Capital Medical University; ⁴Key Laboratory of Neurodegenerative Diseases, Ministry of Education, Beijing, China; ⁵Radiology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Objectives: Mild hypothermia renders potent neuroprotection against acute brain injury.^1,2 Recent reports demonstrate that adenosine 5′-monophosphate (AMP), a natural molecule of ATP catabolism, plays a role in thermoregulation and induces hypothermia in mice.^3,4 This study therefore sought to determine whether AMP induces hypothermia in rats and to study its collective effects on focal cerebral ischemia induced by two-hour MCAO.

Methods: AMP was dissolved in normal saline and intraperitoneally injected into rats. Cerebral ischemia was induced by two-hour middle cerebral artery occlusion followed by reperfusion. ¹ Core body temperature, physiological parameters, blood gas and blood electrolytes were monitored. Western blots were performed to detect AMPK expression and phosphorylation.

Results: Intraperitoneal injection of AMP induced hypothermia in a dose-dependent manner (Figure A). Four mmol/kg AMP induced promising mild hypothermia for 2.5 h. Unexpectedly, AMP-induced hypothermia failed to reduce infarct volume after brain ischemia in rats; instead, it exaggerated the ischemic damage, indicated by an increased infarct volume as well as increased rates of hemorrhagic transformation, seizure and animal death (Figure B and C). Physiological parameter monitoring revealed that AMP causes bradycardia and profound hypotension (Figure D), leading to cerebral hypoperfusion. Furthermore, AMP administration resulted in severe hyperglycemia (Figure E), metabolic acidosis and hypocalcemia. Additionally, western blots demonstrated early dephosphorylation and degradation of AMP-activated kinase in ischemic cortexes in AMP-treated rats (Figure F).

Figure

Conclusions: Our findings suggest that AMP induces hypothermia in rats, probably via limiting cellular access to glucose. However, the potential neuroprotection of AMP-mediated hypothermia against ischemia was overwhelmed by the detrimental effects of hypotension and hyperglycemia, thus making AMP an unlikely agent for inducing hypothermia to protect brain against ischemic injury.

703. A clinically relevant, precise, and noninvasive model of therapeutic hypothermia in the rat

A. Lagina¹, K. Reed², R. Kumar³ and J. Sullivan³

¹Emergency Medicine; ²MS III; ³Emergency Medicine and Physiology, Wayne State University, Detroit, Michigan, USA

Objectives: Hypothermia is neuroprotective after brain ischemia. Therapeutic hypothermia after resuscitation from cardiac arrest is increasingly used in the clinical setting, and may soon find wider clinical application in focal ischemic stroke. Experimental models of hypothermia range from rudimentary (placing the animal in a ‘cold room’) to extremely elaborate. The state-of-the-art is described by DeBow and Colbourne: an implanted brain telemetry system is used to cybernetically control an array of heaters, misters and fans to maintain awake, freely-moving animals at target temperatures. Such systems, while effective and experimentally robust, are extremely costly, and do not model clinical practice. Therapeutic hypothermia is usually instituted via water-cooled extracorporeal systems in conjunction with sedation, and with monitoring of body core temperatures instead of brain temperatures. We undertook the development of a clinically relevant model of therapeutic hypothermia for use in the rat.

Methods: Veterinary warming blankets (Mul-T-Pads) were purchased from Gaymar, resized for the rat, and re-sealed with a heat-sealer (Scotchpak). The pad was fitted to a gel transfer cooling tank (Multi-Temp III, Amersham). Male Sprague-Dawley rats (N = 5) were subjected to 2 h focal brain ischemia under isoflurane/N₂O via MCAO technique. Thermocouples (Physitemp) monitored brain, temporalis and rectal temperatures. Brain thermocouples were stereotactically implanted in the MCA distribution bilaterally. Mean arterial pressure, heart rate and blood glucose were monitored by arterial catheter. Hypothermia was instituted at the onset of reperfusion with an initial tank temperature of 15°C. Animals were sedated with lorazepam during the 4 h hypothermic interval. Animals were reperfused for seven days, and brains were taken to cresyl violet staining to assess stroke volume.

Results: This system proved to be highly responsive and practical, allowing for exquisite brain and core temperature control. Target temperature (32°C rectal) was attained by 35 mins in all animals. Thereafter, core temperature was easily maintained to within 0.5°C with minimal adjustments of tank temperature and blanket position. Differences between ischemic and contralateral brain temperatures were negligible. Brain temperatures were closely approximated by temporalis temperature, even during rewarming, when brain temperatures rose more slowly than core temperatures. Arterial pressure, heart rate and glucose did not undergo significant fluctuations. Animals tolerated the procedure well and survived the 7-day reperfusion interval. Cresyl violet staining demonstrated appropriate placement of thermocouples and decreased stroke volumes compared to ischemic sham-hypothermia controls.

Conclusions: The system described here is an inexpensive, easily constructed and clinically relevant model of therapeutic hypothermia in the rat. Animals can be quickly taken to target temperature and maintained for long intervals. Disadvantages include the inability to use this system in non-sedated, freely-moving animals, although we are compelled to point out that therapeutic hypothermia is not performed in non-sedated, freely-moving patients. The entire apparatus can be constructed for about $3000 (including the tank). As temporalis temperature closely and accurately parallels brain temperature in this model, the use of invasive brain temperature monitoring could be deferred in preclinical studies.

752. Hypothermia alters protein sumoylation during early reperfusion after focal brain ishemia in the rat

J. Sullivan¹, A. Lagina², R. Kumar¹ and J. Wang²

¹Emergency Medicine and Physiology; ²Emergency Medicine, Wayne State University, Detroit, Michigan, USA

Objectives: Recent reports indicate that the Small Ubiquitin-Like Modifier (SUMO) protein participates in extensive protein conjugation (sumoylation) during early reperfusion after brain ischemia, with signal for extensive protein sumoylation present as early as 30 mins of reperfusion. The relative salutary/detrimental effect of this massive sumoylation remains unclear. Moreover, there is evidence that both hypothermia and hibernation topor increase protein sumoylation. We hypothesized that hypothermia administered at the onset of reperfusion after focal brain ischemia would accelerate or accentuate overall protein sumoylation.

Methods: Male Sprague-Dawley rats (N = 3) were subjected to 2 h focal brain ischemia under isoflurane/N₂O using MCAO technique. At the onset of reperfusion, hypothermia was instituted utilizing a water-cooled extracorporeal system consisting of a modified veterinary heating blanket coupled to an Amersham gel cooling apparatus. Body core, brain and temporalis muscle temperatures were monitored throughout the hypothermic interval by thermocouples (Physitemp), and MAP, serum glucose and heart rate were monitored via arterial catheter. At the end of the reperfusion/hypothermic interval, animals were transcardially perfused with ice-cold isotonic saline and the brains rapidly removed. Cortex and striatium in the MCA distribution were taken from both ischemic and contralateral hemispheres and used for gel electrophoresis and Western blotting with antibody directed against SUMO 2/3.

Results: Sham-operated control animals demonstrated no protein sumoylation in any brain region. Animals subjected to 2 h of brain ischemia and 2 h of normothermic reperfusion demonstrated heavy protein sumoylation in both cortex and striatum, but no sumoylation in contralateral brain. Animals subjected to 2 h of brain ischemia and 2 h of hypothermic reperfusion demonstrated markedly decreased protein sumoylation compared with ischemic animals.

Conclusions: These results suggests that hypothermia significantly alters the pattern and timing of protein sumoylation in ischemic brain when instituted at the onset of reperfusion after MCAO. Detailed time course studies are underway, as well as experiments to determine whether ischemia-induced sumoylation is a truly neuroprotective phenomenon and, if so, which sumoylated proteins mediate any effect on neuronal survival.

784. An evaluation of the safety of 3 weeks of focal cortical hypothermia in rat

A. Auriat¹, M. Penner¹, G. Silasi², D. Clark² and F. Colbourne^1,2

¹Psychology; ²Centre for Neuroscience, University of Alberta, Edmonton, AB, Canada

Objectives: Hypothermia is neuroprotective following global and focal cerebral ischemia. Cooling is frequently limited to a short period during the acute phase of injury (e.g., 48 h treatment), but several types of brain injury produce cell death over a longer period and edema can persist for many days. Thus, extended cooling might provide additional benefit. However, before treatment efficacy can be addressed the safety of long-term cooling must be determined.

Methods: Rats were cooled with a metal cooling strip placed on the skull overlying the motor cortex in the right hemisphere. This strip was cooled by flushing it with cold water via a tethered system. ¹ Cooling was quickly initiated and maintained at a mildly hypothermic level for 3 weeks prior to gradual re-warming over 24 h. Control rats were treated similarly, but not cooled. In part 1, several physiological parameters were measured including blood pressure, heart rate, body and cortex temperature (n = 2). In part 2, brains were assessed for histological damage (hematoxylin and eosin (H&E) and fluoro jade B) 3 days after control (n = 8) or hypothermia treatment (n = 8) ended. In part 3, Golgi-Cox staining was used to assess neuronal complexity 3 days after re-warming (n = 8) or at the same time in control rats (n = 8). Finally, in part 4 electron microscopy was used to look for ultrastructural signs of tissue abnormalities in cooled and control brain. In several of these experiments we looked for behavioral abnormalities at 3 days after cooling or control treatment ceased. The corner turn, cylinder and horizontal ladder tests were used.

Results: Part 1: None of the physiological parameters were significantly affected by cooling. Brain temperature was lowered by 3–4°C for the duration of cooling. Part 2: No cell death was labeled with fluoro jade B in either cooled or control brains. H&E staining produced some dark neurons, however, these cells were present in both cooled and control hemispheres of cooled rats as well as in controls. No other indications of significant histological damage were present. The Golgi-Cox and electron microscopy data are yet to be completely analyzed. However, behavioral data indicate that no asymmetries or deficits occur with 3 weeks of focal brain cooling.

Conclusions: Studies have consistently shown great protection with longer bouts of systemic hypothermia (e.g., 48 versus 12 h). ² We have observed similar effects with focal cooling after ischemic stroke. The current findings suggest that local brain cooling may be administered for a prolonged period (3 weeks) without any significant physiological, anatomical or behavioral side effects.

787. A comparison of 12 and 48 h of selective brain hypothermia after permanent focal ischemia in rat

D. Clark¹, M. Penner², S. Wowk² and F. Colbourne^1,2

¹Neuroscience; ²Psychology, University of Alberta, Edmonton, AB, Canada

Objectives: Hypothermia provides significant neuroprotection in rodent models of ischemic brain damage. Indeed, we recently showed a duration-dependent treatment effect such that 48 h of systemic hypothermia provided greater histological and behavioral protection at 7 days after onset of permanent focal ischemia. ¹ However, the ideal treatment parameters and method of inducing hypothermia has not been determined. It is also important to evaluate whether treatments provide enduring protection. Thus, we examined the long-term impact of 12 and 48 h of hypothermia treatment (33°C, 1 h delay) on outcome after permanent middle cerebral artery occlusion (pMCAO). Importantly, we used a novel method of inducing focal brain hypothermia developed in our lab. ²

Methods: Rats were trained on a skilled reaching task and evaluated for baseline neurological status and walking ability. Stroke was induced by cauterization of the distal middle cerebral artery. Rats were randomly assigned to remain normothermic or receive focal hypothermia treatment one hour following occlusion for either 12 or 48 h as described previously. ² Rats were cooled at 2°C/h and rewarmed slowly at 1°C/h. Walking ability was measured on days 7 and 28 after stroke onset. Neurological deficits were measured on day 7 while skilled reaching ability was evaluated over 5 days starting one month following stroke onset. Animals were then euthanized to determine lesion volume.

Results: The 48 h treatment (versus control normothermic rats) significantly improved skilled reaching and walking ability while neurological impairment was reduced. No significant benefit was observed with the 12 h cooling protocol. Histological analysis is ongoing and will be presented at the meeting.

Conclusions: Our results thus far indicate that delayed brain-selective hypothermia provides significant long-term behavioral protection in rats experiencing a pMCAO. Similar to our previous findings with systemic cooling, ¹ there is a clear duration-dependent effect. These findings suggest that either brain-selective or systemic cooling treatments, if prolonged, will improve outcome following ischemic stroke.

789. Treatment of intracerebral hemorrhage in rats with 12 h, 3 days and 6 days of selective brain hypothermia

M. Penner¹, M. Fingas², G. Silasi² and F. Colbourne^1,2

¹Psychology; ²Centre for Neuroscience, University of Alberta, Edmonton, AB, Canada

Objectives: Intracerebral hemorrhage (ICH) is a devastating stroke, which accounts for 10% to 20% of all strokes. There is no proven treatment to reduce morbidity and mortality. In this study, we wanted to assess whether focally cooling the injured hemisphere following ICH would be beneficial. We also wanted to vary the duration of hypothermia to assess whether prolonged cooling is more beneficial than brief cooling.

Methods: We caused an ICH in rats by injecting 100 μL of autologous blood into the striatum. Systemic hypothermia has reduced blood-brain-barrier disruption, inflammation and edema, but has not consistently improved behavioral or histological outcome after ICH in animal studies. As this might relate to the choice of cooling method, we used a method that selectively cools the injured hemisphere to ∼32°C (striatum) while the body remained normothermic. Cooling was initiated 1 h after ICH and lasted for 12 h, 3, or 6 days, and was followed by slow rewarming (∼1°C/h). These groups were compared to a control group, which remained normothermic. We assessed functional outcome during weeks 2 and 3 post-ICH, and euthanized the rats on day 21 post-ICH. We used several behavioral tasks to assess general impairment (neurological deficit scale), walking ability (grid walk test), forelimb asymmetry (cylinder), and reaching ability (tray reaching task). We also assessed lesion volume at the end of the experiment (day 21 post-ICH).

Results: The ICH caused significant impairments in all of the behavioral tasks. The longest hypothermia treatment (6 days) significantly reduced forelimb asymmetry for both post-lesion assessments. However, none of the treatments significantly improved outcome on any of the other behavioral tasks. In addition, there was no significant reduction in lesion volume in any of the groups.

Conclusions: These results, as well as previous studies ¹ indicate that hypothermia is not a substantial neuroprotectant following ICH. However, it may still improve functional recovery, as well as reduce life-threatening edema.

814. Focal hypothermia by epidural cooling provides brain protection in porcine model of middle cerebral artery occlusion

H. Cheng

Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, China

Objectives: Hypothermia has been shown to be neuroprotective in many animal models and several human trials of brain ischemia. However the protective effect against ischemic stroke by systemic hypothermia is limited by the cooling rate and it has severe complications. Our perior researchs show that the selective brian hypothermia (moderate and deep brian temperature) by epidural space cooling can be induced rapidly, and may be a useful strategy for reducing infarct volume after the onset fo ischemia. This study was designed to observe the histological morphology and differences of apoptosis on the brain tissue in a swine model of permanent middle cerebral artery occlusion (PMCAO) and eveluated the effect of protection by epidural cooling.

Methods: PMCAO was performed in 12 domestic swine assigned to groups A and B. In group A, the cranial and rectal temperatures were maintained at normal range for six hours after PMCAO. In group B, Cranial temperature was reduced to moderate (deep brain) and deep (brain surface) temperature and maintained at the level for 5 h following one hour after PMCAO, by the method of epidural cooling. Epidural cooling was performed using cold-saline (4 degrees C) perfusion into the epidural space through a flexible double-lumen catheter. The dripping speed of cold saline was controlled to maintain the target temperature. All animals were euthanized 6 h after MCAO; brain sections were perfusion-fixed and cryopreserved. Hematoxylin and eosin histology were used to characterize cell damage morphologically. Neuronal apoptosis were characterized by TUNEL histochemistry.

Results: Following the epidural cooling, the target temperatures were easily controlled (from mild to deep hypothermia on focal brain region). The body temperature was maintained at normal range within 6 perfusion hours. The structure of brain tissue in the infarcted regions maintained more intact, and TUNEL-positive cells were significantly fewer in the hypothermic group than in the normal temperature group.

Conclusions: The present study has demonstrated, with histological confirmation and apoptosis mechanisms, that epidural cooling may be a useful strategy for neuroprotection after the onset of ischemia.