Design

A randomized, controlled, animal study.

Time and setting

A total of seven healthy adult rhesus monkeys, of both genders, weighing 9.2 ± 2.6 kg, were purchased from the Experimental Center of Kunming Institute of Zoology (license No. SYXK (Dian) 2005-0004). The experiments were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China^[22].

Methods

Establishment of selective cerebral deep hypothermia and blood flow occlusion model

After anesthesia, mean arterial blood pressure was monitored by intubation of the proximal right inguinal artery during hypothermic perfusion, while intubation of the proximal left inguinal vein was used for transfusion of systemic circulation blood flow after rewarming. The right internal carotid artery and both internal carotid veins were clamped by an aneurysm blocking clamp and connected to a circulation pump (blood pump, DKP-22, NIKKISO, Japan). An ultrafilter (F-60, FRESENIUS, Germany) and water bank (SARNS TCM, ANN ARBOR MICHINA, USA) were used to build local extracorporeal circulation. Central venous pressure was monitored by intubation of the proximal right internal jugular vein. A pin-shaped brain temperature sensor was inserted into the right frontal lobe and connected to a brain temperature monitoring system (TH-5; Department of Medical Engineering, The University of Virginia, VA, USA). General heparinization was conducted prior to temperature reduction by intravenous injection of      50 IU/kg heparin.

The bilateral external jugular veins, the left common carotid artery and the internal jugular vein were clamped in the two-vessel occlusion group, and the bilateral vertebral arteries were also clamped in the four-vessel occlusion group. After ischemia for 10 minutes at common temperature, Ringer's solution (Shanghai Baite Medical Product Co., Ltd., Shanghai, China) at 4°C was infused via the right internal carotid artery, at 10 mL/kg per minute. In addition, perfusion solution was refluxed through the right internal jugular vein, and systemic circulation blood was refluxed through the right inguinal vein. Redundant water was removed through the ultrafilter (F-60, Fresenius, Germany) and the blood was rewarmed to 38°C and transfused into the left inguinal vein. After the brain temperature was reduced to ≤18°C, the perfusion rate was reduced or interrupted, at 2–10 mL/kg per minute to maintain the brain temperature at ≤18°C. Hypothermic perfusion was terminated after 60 minutes, and the right internal carotid artery and vein were unclamped to restore the brain temperature to 36°C^{[5, 7]}. The monkeys exhibited stable vital signs after surgery and recovered from surgery within 24 hours. They could move and take food normally, and their visual acuity was normal.

Microdialysis sample collection and processing

A microdialysis probe (dialysis-membrane effective length 4 mm, molecular weight cutoff 20 ku) was implanted into the right frontal lobe (the probe was implanted into the right frontal lobe and brain tissues from different sites were harvested for pathological detection). The right frontal lobe was slowly microdialysed using a micro- injection pump at       2.5 μL/min 90 minutes prior to blood flow blocking, and dialyzate was collected at 60 minutes. Extracellular fluid was collected prior to ischemia, after ischemia for 10 minutes, after hypothermic perfusion for 60 minutes and after rewarming for 40 minutes, with one tube (100 μL) every 20 minutes for seven tubes in total. The tubes were stored at –70°C until amino acid analysis was performed.

Derivatization reactions

Dialyzate (50 μL) was harvested, mixed with drying agent (absolute alcohol: triethylamine: water = 2:2:1) for 3 minutes, dried in a 60–100 Mt vacuum, mixed with 100 μL of freshly prepared derivatized reagents (alcohol: water: triethylamine:phenyl isothiocyanate=7:1:1:1), and shaken for 5 minutes for derivation in a dark box for    10 minutes. The products were dried in vacuum for 15 minutes, mixed with 100 μL of dilution solution (Na₂HPO₄ 710 mg/L, 10% H₃PO₄, pH 7.40, followed by acetonitrile to a final content of 5%), and filtered using a syringe filter (0.45 μm). Filtrate (20 μL) was harvested and sampled.

Detection of Glu content in the brain

The Glu chromatographic peak from various samples was detected by chromatography (Waters 510 pump, MA, USA), and the retention time of unknown amino acid peaks was qualified and quantified according to the retention time of standard amino acid peaks. The chromatographic peak area of four amino acid samples was calculated using high performance liquid chromatography-ultraviolet detection^[23] with the formula:   C = R1/R2 × D × N; R1, peak area of detected samples; R2, peak area of amino acid standard sample; D, concentration of amino acid standard sample (μM); N, dilution times; C, concentration of detected sample (μM).

Ultrastructural observation of brain tissues

Bilateral brain tissues of monkeys were cut into tissue blocks of 1 mm³, fixed with glutaral (3%)-osmium tetroxide (10 g/L), dehydrated with 30–100% acetone, embedded with epoxy resin, coronally sectioned using LKV-V ultramicrotome (50 nm thick), stained with uranyl acetate-lead citrate, and observed under a JEM-100CX transmission electron microscope (Tokyo, Japan) to detect the structure of the cell membrane, nuclear membrane, cytoplasm, mitochondria and endoplasmic reticulum.

Statistical analysis

Data were analyzed using SPSS 12.0 software (SPSS, Chicago, IL, USA) and expressed as mean ± SD. Intergroup differences in Glu content at different time points were compared using one-way analysis of variance. A value of P < 0.05 was considered statistically significant.

(1) Selective deep hypothermia resuscitation after severe ischemia in monkey brain stimulated release of excitatory neurotransmitters such as glutamic acid and inhibited cytotoxicity, but did not damage cell ultrastructure in brain tissues.
(2) Selective deep hypothermia enhanced brain tolerance to ischemia and hypoxia.

1. 猴脑严重缺血后行选择性深低温复苏，可使谷氨酸等兴奋性神经递质释放并抑制细胞毒性作用，而猴脑组织细胞超微结构无明显损害。
2. 选择性深低温能增加脑对缺血缺氧的耐受性。

实验设计构思介绍:
猴脑选择性深低温断血流能增加脑对缺血、缺氧的耐受性，减少心肺并发症，脑缺血后兴奋性氨基酸大量释放所导致的‘兴奋毒性’是缺血性脑损伤和神经细胞死亡的主要机制之一；故实验对选择性深低温断血流复苏前后不同时点的脑兴奋性氨基酸的变化进行动态观察，并结合脑超微结构观察进行验证。