Therapeutic Window of Selective Profound Cerebral Hypothermia for Resuscitation of Severe Cerebral Ischemia in Primates

Abstract

It is well recognized that brain death starts to occur just 4–6 min after cardiac arrest, and few attempts at resuscitation succeed after 10 min of severe cerebral ischemia and anoxia. We sought to determine the therapeutic window of selective cerebral profound hypothermia of primates following severe cerebral ischemia in primates. Fourteen rhesus monkeys with severe cerebral ischemia were divided into four groups: normothermia (n = 3); profound hypothermia I (n = 4), with cooling initiated 10 min after ischemia; profound hypothermia II (n = 4), with cooling initiated 15 min after ischemia; and profound hypothermia III (n = 3), with cooling initiated 20 min after ischemia. Severe cerebral ischemia was induced by clamping both the internal and external carotid arteries, as well as the internal and external jugular veins. Profound cerebral hypothermia (15.8° ± 0.9°C) was achieved and maintained for 60 min, and the animals were then re-warmed gradually. All four animals in hypothermia group I survived without any neurological deficits. Only 1 animal survived and 3 animals died in hypothermia group II. All 4 animals died in both hypothermia group III and the normothermia group. Neurological functions were normal in all surviving animals, and MRI scans showed no cerebral infarction in these animals. Microscopic examination showed no injured neurons in the hippocampus and cerebral cortex of the surviving animals, and showed that the heart, lung, liver, and kidneys were normal in these animals. Our data indicate that post-ischemic profound cerebral hypothermia provided significant cerebral protection with no systemic complications, and that the effective therapeutic window is more than 10 min, but less than 15 min, after severe cerebral ischemia.

Introduction

I t is well recognized that brain death starts to occur 4–6 min after cardiac arrest, and few attempts at resuscitation succeed after 10 min of severe cerebral ischemia and anoxia. Mild to moderate hypothermia (33–35°C) has been found to play an important role in emergency brain resuscitation of patients with cardiac arrest (The Hypothermia After Cardiac Arrest Study Group, 2002). Profound hypothermia may provide more cerebral protection; however, systemic profound hypothermia may cause serious cardiovascular and pulmonary complications (Kochanek and Safar, 2003). A technique to selectively cool the brain without induction of systemic hypothermia may provide similar cerebral protection, but without systemic complications, and may thus be advantageous for brain resuscitation (Jiang et al., 2006).

In 1996 Schwartz and colleagues reported that isolated profound cerebral hypothermia (19°C for 30 min) could be achieved by perfusion of autologous cooled blood through the common carotid artery, and when initiated after cerebral ischemia in baboons, all the animals survived without neurological deficits (Schwartz et al., 1996). Furthermore, we recently reported that selective profound cerebral hypothermia (15.5°C for 60 min) was achieved by perfusion of cooled Ringer's solution via the right internal carotid artery, and that when initiated at the same time as cerebral ischemia in primates, all the animals survived with no neurological deficits (Jiang et al., 2006). However, there are no reports that explore whether primates will survive if selective profound cerebral hypothermia is induced after complete cerebral ischemia. We sought to determine the therapeutic window of post-ischemic profound cerebral hypothermia for resuscitation of primates with severe cerebral ischemia.

Methods

Animal groups

All procedures were approved by the ethics committee of the institutional animal care and use committee of the Chinese Academy of Science. Fourteen rhesus monkeys (weight 6–10 kg) received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Committee of Medical Research, and the “Guide for the Use of Laboratory Animals” issued by the institute of Laboratory Animal Resources of China. Fourteen rhesus monkeys with severe cerebral ischemia were divided into four groups: normothermia (n = 3); profound hypothermia I (n = 4), with cooling initiated 10 min after ischemia; profound hypothermia II (n = 4), with cooling initiated 15 min after ischemia; and profound hypothermia III (n = 3), with cooling initiated 20 min after ischemia.

Severe cerebral ischemia and selective profound cerebral hypothermia

Anesthesia was induced with ketamine hydrochloride (10 mg/kg) administered intramuscularly. Thereafter, the trachea was intubated and ventilation controlled. Anesthesia continued with isoflurane (1.5–2.0%) in oxygen. A triple-lumen catheter was inserted into the inferior vena cava via the right femoral vein to monitor central venous pressure and for venous access. The right femoral artery was cannulated to monitor mean blood pressure (BP), arterial blood gas, serum electrolytes, and hematocrit. Throughout the experiment, electrocardiography, arterial blood pressure, and central venous pressure were continuously recorded. Cranial burr holes were made bilaterally and temperature probes (model HYP-O33-1-T-G-60-SMP-M; Omega Engineering, Inc., Stamford, CT) were inserted into the parenchyma of the frontal lobe and connected to an electronic thermometer (model #DP81T; Omega Engineering, Inc.) to record brain temperature, as previously reported (Jiang et al., 1991). Rectal temperatures were also continuously recorded (model TH-5; Physitemp, Inc., Clifton, NJ). The animals were placed on an electric heating blanket to maintain body temperature (Table 1).

Table 1.

Brain and Rectal Temperatures (°C) in the Four Study Groups

	Normothermia		Hypothermia I		Hypothermia II		Hypothermia III
Time	Brain	Rectal	Brain	Rectal	Brain	Rectal	Brain	Rectal
Pre-perfusion	36.7 ± 0.5	37.5 ± 0.8	36.9 ± 0.4	37.3 ± 0.6	36.9 ± 0.5	37.6 ± 0.8	36.6 ± 0.5	37.3 ± 0.9
During perfusion
10 min	36.6 ± 0.4	37.6 ± 1.0	15.9 ± 0.7	33.3 ± 1.1	15.6 ± 1.2	32.8 ± 1.1	15.9 ± 1.4	32.7 ± 1.6
20 min	36.5 ± 0.6	37.7 ± 0.9	15.6 ± 1.1	32.8 ± 1.0	15.8 ± 1.0	33.1 ± 1.0	15.7 ± 1.5	33.1 ± 1.7
30 min	36.5 ± 0.5	37.6 ± 0.8	16.0 ± 1.2	33.1 ± 0.9	15.7 ± 1.1	33.3 ± 1.2	15.4 ± 1.4	33.0 ± 1.6
40 min	36.6 ± 0.6	37.5 ± 0.7	16.1 ± 1.3	33.0 ± 0.9	15.8 ± 0.9	32.9 ± 1.0	15.6 ± 1.3	32.9 ± 1.8
50 min	36.7 ± 0.5	37.6 ± 0.8	15.6 ± 0.9	32.8 ± 1.1	16.2 ± 1.2	33.0 ± 1.1	16.0 ± 1.3	33.1 ± 1.7
60 min	36.6 ± 0.5	37.5 ± 0.9	15.7 ± 0.8	33.2 ± 1.2	15.7 ± 1.3	32.9 ± 0.9	16.2 ± 1.2	33.3 ± 1.7
Post-perfusion
10 min	36.3 ± 0.9	37.2 ± 1.0	27.7 ± 1.5	34.1 ± 1.5	26.6 ± 1.9	34.0 ± 1.7	28.1 ± 1.5	34.3 ± 1.6
20 min	36.3 ± 0.7	36.9 ± 1.0	31.9 ± 2.1	34.7 ± 1.4	30.1 ± 2.1	34.9 ± 1.5	31.2 ± 1.9	34.7 ± 1.5
30 min	36.4 ± 0.8	36.9 ± 1.1	32.7 ± 1.8	35.3 ± 1.6	33.3 ± 1.7	35.6 ± 1.8	33.0 ± 1.6	35.3 ± 1.6
40 min	36.2 ± 0.7	37.0 ± 1.2	33.5 ± 1.9	35.5 ± 1.5	34.2 ± 1.6	35.7 ± 1.6	34.3 ± 1.7	35.5 ± 1.7

Severe cerebral ischemia was induced and selective profound cerebral hypothermia (15.8° ± 0.9°C) was achieved and maintained for 60 min as previously described (Jiang et al., 2006). Briefly, the carotid arteries and jugular veins were exposed bilaterally in the neck. A catheter was inserted into the right internal carotid artery for infusing cold Ringer's lactate solution into the brain. Both internal jugular veins were cannulated and connected to a centrifugal pump so hemodilution and recirculation of cold Ringer's lactate solution could be achieved. After systemic heparinization (100 U/kg), the left internal carotid artery, both external carotid arteries, and both external jugular veins were temporarily clamped. At the same time, cooled Ringer's lactate solution (4°C) was infused through the right internal carotid artery and flowed out of the brain though both internal jugular veins in this closed-circuit system. The cooled solution lowered the cerebral temperature to the target temperature (15.8° ± 0.9°C) within 10 min, and it was maintained for 60 min with the infusion rate ranging from 20–40 mL/min to maintain brain temperature at the target value. Body temperature was maintained at 33.1° ± 0.67°C. Thereafter, the pump flow was shut off, the brains were rewarmed spontaneously, and the carotid arteries and jugular veins were opened to resume normal cerebral blood circulation. After cerebral temperature increased to >34°C, the femoral and carotid catheters were removed. In the normothermia control group, Ringer's solution at 37°C was infused in the same manner in three monkeys.

Neurological function assessment and MRI scanning of surviving animals

The animals in hypothermia group I were examined daily for neurological function using the method outlined by Spetzler and colleagues, a 100-point scale that evaluated motor function, behavior, cranial nerve function, and ocular function (Spetzler et al., 1980). MRI scans (1.5 tesla; Phillips Co., The Netherlands) were taken before ischemia and 3 weeks after ischemia. MRI scans were not done in the animals in the hypothermia II and III groups due to their poor condition, and they died shortly after of ischemic stroke.

Brain tissue collection and sectioning

The surviving animals were sacrificed humanely with a lethal dose of pentobarbital 3 months later. All animals were infused via both carotid arteries with FAM (a mixture of 40% formaldehyde, glacial acetic acid, and methanol, 1:1:8 by volume) for 20 min at a pressure of 100–120 mm Hg following a 5-min perfusion with physiological saline. The hippocampus was removed and stored overnight in 4% paraformaldehyde at 4°C. Coronal sections were cut 50 μm thick with a vibratome (Leica VT 1000S; Leica, Germany) and stained with hematoxylin and eosin (H&E). The heart, lung, liver, and kidneys were removed and embedded in paraffin, sectioned (10 μm thick), and stained with H&E.

Results

Arterial blood gas values (pH, PaO₂, and PaCO₂) were normal at pre-perfusion, during perfusion, and at post-perfusion in the four groups (Table 2).

Table 2.

Blood Gas Values in the Four Study Groups

	Pre-perfusion			During perfusion			Post-perfusion
Group	pH	PaO₂ (mm Hg)	PaCO₂ (mm Hg)	pH	PaO₂ (mm Hg)	PaCO₂ (mm Hg)	pH	PaO₂ (mm Hg)	PaCO₂ (mm Hg)
Normothermia	7.40 ± 0.06	301 ± 40	41 ± 4	7.37 ± 0.06	321 ± 34	37 ± 6	7.43 ± 0.06	309 ± 34	39 ± 5
Hypothermia I	7.37 ± 0.05	320 ± 32	37 ± 5	7.35 ± 0.11	312 ± 29	43 ± 5	7.35 ± 0.10	323 ± 25	44 ± 6
Hypothermia II	7.35 ± 0.04	312 ± 29	43 ± 3	7.42 ± 0.10	331 ± 39	40 ± 7	7.42 ± 0.12	328 ± 31	41 ± 5
Hypothermia III	7.42 ± 0.07	324 ± 28	40 ± 5	7.44 ± 0.13	302 ± 35	36 ± 7	7.37 ± 0.13	311 ± 37	37 ± 4

All four animals in hypothermia group I survived with no neurological deficits. Only one animal survived and the other three animals died at 12, 16, and 20 h after ischemia in hypothermia group II. All three animals died at 5, 9, and 12 h after ischemia in hypothermia group III. All three animals died at 3, 5, and 6 h after cerebral ischemia in the normothermia group.

All the animals in hypothermia group I awakened from anesthesia neurologically intact, with a score of 100 on the first day after surgery and thereafter. MRI scans did not show any cerebral infarction or ischemic changes at 3 weeks after ischemia in surviving animals (Fig. 1).

FIG. 1.

MRI scans of monkey brains (A) before cerebral ischemia, (B) during cooling, and (C and D) 3 weeks after cerebral ischemia in surviving animals.

Microscopic examination did not reveal any injured neurons in the hippocampus, CA1, or the parietal cortex in surviving or non-surviving primates (Fig. 2). Microscopic examination also showed that the heart, lung, liver, and kidneys were normal in all of the animals (Fig. 3).

FIG. 2.

Microscopic examination did not detect any injured neurons in the parietal cortex (A) or hippocampus CA1 area (B) of the surviving primates, or the parietal cortex (C) or hippocampus CA1 area (D) in non-surviving primates (H&E 100 × ). Furthermore, examination at higher magnification also did not reveal any injured neurons in the parietal cortex (E) or hippocampus CA1 (F) in any of the animals (H&E 200×).

FIG. 3.

Microscopic examination did not show any pathological changes in the heart (A), lung (B), liver (C), or kidney (D) in any of the animals (H&E 100×).

Discussion

We found that post-ischemic profound cerebral hypothermia provides significant cerebral protection with no systemic complications, and that the effective therapeutic window is longer than 10 min, but less than 15 min, which indicates that post-ischemic profound hypothermia may be useful for resuscitation of patients after cardiac arrest. However, we cannot rule out the possibility that the lack of protection seen in the 15- and 20-min delay groups may have been due at least in part to the overall longer duration of cerebral ischemia in these groups (75 and 80 min, versus 60 min in the normothermia group).

Profound hypothermia has gained widespread acceptance as an effective technique for brain protection during cardiothoracic surgery (Ananiadou et al., 1992). Total circulatory arrest with body temperature reduced to <20°C (<60 min duration) has become the preferred strategy for the repair of thoracic aortic aneurysms. More recently, Ananiadou and colleagues compared the effects of different degrees of profound hypothermia on neural damage following hypothermic circulatory arrest (75 min), and found that cerebral protection was better at 10°C than at 18°C, indicating that lower temperatures may provide better protection following cerebral ischemia (Ananiadou et al., 1992). However, experimental studies and clinical trials using systemic profound hypothermia for brain protection have been complicated by the injurious systemic effects of total body cooling, including low cardiac output, elevated systemic vascular resistance, ventricular fibrillation, pulmonary edema, and postoperative hemorrhage secondary to heparinization (Kochanek and Safar, 2003; Steen et al., 1980). Thus, a technique to selectively cool the brain without induction of systemic hypothermia may not only provide similar cerebral protection, but may also prevent systemic complications (Jiang et al., 2006). Schwartz and associates reported that isolated profound cerebral hypothermia (19°C for 30 min), initiated at same time as cerebral ischemia in baboons, resulted in all animals surviving with no neurological deficits (Schwartz et al., 1996). We recently reported that selective profound cerebral hypothermia (15.5°C for 60 min), begun at the same time as cerebral ischemia in primates, also resulted in all the animals surviving with no neurological deficits (Jiang et al., 2006). In this study, we found that post-ischemic profound cerebral hypothermia provided significant cerebral protection with no any systemic complications, and that the effective therapeutic window was longer than 10 min, but less than 15 min.

Reduction of cerebral oxygen consumption may be a major mechanism behind the cerebral protection induced by hypothermia. Cerebral oxygen consumption is known to decrease by 6–7% for each degree of temperature reduction during hypothermia. In this study, the brain temperature decreased to approximately 16°C, which would theoretically reduce cerebral oxygen consumption to approximately 0%. Steen and colleagues reported that cardiac output and whole-body oxygen consumption decreased to 52% and 42% of control values, respectively, when dogs' body temperatures were decreased to 29°C by application of external ice packs. Thereafter, cardiac output and oxygen consumption continued to decline progressively until 24 h, when they reached 7% and 28% of control respectively (Steen et al., 1980). More recently, we have found differential gene expression in the hippocampus in the rat following complete cerebral ischemia with treatment using profound hypothermia versus that seen with normothermia. Expression profiles of a total of 75 transcripts in the profound hypothermia ischemia group were statistically significantly different from those seen in the normothermia ischemia group, with 33 of them significantly upregulated, and 42 significantly downregulated. These data suggest that profound hypothermia may have a significant effect on gene expression profiles following temporary global ischemia, and may be part of the mechanism of the cerebral protection afforded by profound hypothermia (Qin et al., 2008); however, these mechanisms require further investigation.

Footnotes

Acknowledgments

This work was supported by grants from the National Key Basic Research Project (no. 2005CB522604), National Health Science (no. 200802093), the Science and Technology Committee of Shanghai (no. 07JC14038 and 08411951900), and the Program for Shanghai Outstanding Medical Academic Leaders.

Author Disclosure Statement

No conflicting financial interests exist.

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