Abstract
In the present study, we investigated the effect of a cerebral protective solution on prolongation of cerebral dormancy time in a rabbit model of occlusion-reperfusion. In a control group, rabbits were anesthetized and the four cerebral arteries (the left and right common carotid arteries and vertebral arteries) were occluded for 7.5 min followed by reperfusion. All six rabbits in the control group died. In contrast, a second group underwent perfusion of a cerebral protective solution for 15 min between artery occlusion and reperfusion. All six rabbits in this group survived. However, when the perfusion solution was changed to 5% glucose solution or rabbit plasma in two other groups, the rabbits in both the latter two groups also died. Neuroprotection was also observed when the protective solution was administered for 30-60 min after the onset of artery occlusion and before the return of blood flow (reperfusion). To understand the high rate of thrombotic stroke in the clinic, we assessed the influence of different organ tissue infusions on blood coagulation in vitro and found that blood clotting occurred faster in the presence of brain tissue infusion compared to liver, kidney, and heart tissue infusions. These results indicate a higher rate of thrombosis in brain tissue compared to any of the other tissues tested. The current study shows that perfusion of a cerebral protective solution produced a significant neuroprotective benefit in our rabbit model of occlusion-reperfusion, suggesting that administration of a cerebral protective solution may be an effective approach for the treatment of ischemic stroke.
The brain is sensitive to ischemia and hypoxia, and traditionally it is believed that irreversible death occurs when there is a lack of blood supply for three to five minutes. According to the gold standard for diagnosis of brain death in the United States, brain death is diagnosed when no blood perfusion is shown by cerebral angiography of four cerebral arteries (the left and right common carotid arteries and the left and right vertebral arteries) and concurrently the presence of a flat electroencephalogram (EEG). However, recent research shows that it is not real brain death which has occurred even when the brain lacks blood supply and loses its function (1, 2) and the brain shows no electrical activity on EEG (3-5). Rather, these changes reflect a dormant state of brain cells due to lack of blood supply and changes in the internal environment. When the normal environmental conditions are restored, the brain cells can be re-activated and can return to their normal functions. We refer to the functional incapacitation caused by ischemia and hypoxia of the brain as ‘cerebral hibernation’ and the period of time from the beginning of this dormant period to the return of cellular function as ‘cerebral dormancy’, and the irreversible loss of brain function as ‘brain death’.
To explore an effective method of restoring normal cellular function after cerebral dormancy, we designed and established a controllable experimental rabbit model of cerebral occlusion-reperfusion and compared the effects of three different solutions (a mixed cerebral protective solution, 5% glucose solution, and rabbit plasma) on the recovery of cellular function after cerebral dormancy. We showed in this study that perfusion of the cerebral protective solution produced significant benefits of neuroprotection in our rabbit model of occlusion-reperfusion.
Materials and Methods
Reagents and apparatus. MS 2000 computer biological signal recording system, pressure transducer, Thumbtack electrode and deep artery blood flow blocking casing were purchased from Sigma–Aldrich (St. Louis, MO, USA). Four-way artery catheter, diazepam injection and 1% heparin anticoagulant were purchased from Invitrogen–Gibco (Carlsbad, CA, USA). Rabbit plasma and 5% glucose solution were obtained from Sunshine Biotechnology (Nanjing, China). Sodium citrate solution (0.1 M), 1/40 M calcium chloride solution, and physiological saline were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The mixed rabbit brain protection solution contained the following per 100 ml: 0.375 g of sodium chloride, sodium lactate 0.0775 g, potassium chloride 0.0075 g, calcium chloride 0.005 g, 2.5 g of glucose and dextrane 7.5 g. All other chemicals used were of the highest commercial grade available.
Experimental animals and breeding. In total, 36 healthy rabbits (18 male and 18 female) were purchased from Guangxi Animal Center (Nanning, China). The rabbits, 2 years of age and 3-3.5 kg each, were raised at the Experimental Animal Center of Guangxi University of Chinese Medicine under specific pathogen-free conditions and were randomized into groups A, B, C, D, E, F with 6 rabbits in each group. The rabbits were cared for in accordance with the guidelines for the treatment of experimental animals published by the Ministry of Science and Technology of the People's Republic of China in 2006. This study was carried-out in strict accordance with the recommendations set forth in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (eighth version, 2010). The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Guangxi University of Chinese Medicine (Nanning, China).
MS 2000 computer biological signal records analysis. The incoming line of the EEG recording electrode was connected to the leading electrode and a channel socket was inserted. The blood pressure transducer was filled with heparin anti-coagulant and inserted into the two-channel interface. The MS 2000 system was started, with “electrical brain” chosen in the first channel selection and “pressure” in the second. The pressure signal was calibrated and a continuous oscilloscope display was chosen.
Establishment of the experimental rabbit model. Each rabbit received 10 mg/kg of anesthetic via intravenous injection. The hair on the neck was removed, a midline subcutaneous incision was extended for 6 to 8 cm, the muscles were separated by blunt dissection, and the trachea and arteries were exposed.
The vertebral arteries were separated bilaterally, and an arterial occlusion sleeve was placed on each artery. Next, the cervical fascia along one side of the common carotid artery was separated from the clavicle to the prevertebral fascial layer with hemostatic forceps. We continued blunt dissection of the prevertebral fascia in the initial segment of vertebral artery where pulsation of the carotid artery is apparent in order to expose the vertebral artery. We then carefully threaded a silk thread at the bottom of the arteries with curved ophthalmic forceps, and held up the two ends of the silk thread with two fuses placed through the artery, thereby blocking blood flow. The same procedure was applied to the opposite side to occlude blood flow.
We separated both sides of the common carotid artery and connected a four-way artery catheter. We separated the carotid arteries bilaterally from the trachea with blunt dissection. We threaded two silk threads to preserve the lower segments of the arteries, and cut the common carotid arteries in the middle after occluding the arteries distally and proximally, leaving a 3-cm segment. A four-way catheter was inserted at the location of the cut segment into the artery bilaterally. The four-way catheter was fixed with silk threads. The distal end of the catheter was connected to transfusion tubes, while the proximal end was connected to the blood pressure sensor. The valve was opened, thereby linking the blood pressure sensor to the great vessels of the heart and providing us with the ability to measure arterial pressure. The distal end of the catheter was connected to transfusion tubing. In the same way, the two ends of three-way artery were inserted into the other side of the common carotid artery and connected. The distal end of the catheter was connected to transfusion tubes, and the carotid artery blood flow was opened.
To connect the electrical leads to the rabbit, a cut was made and the skull exposed. We drilled a small hole 2-3 mm from the sagittal suture and 5-6 mm in front of the herringbone seam, and connected a thumbtack guiding electrode at this location. The reference electrode was placed in the skin incision.
Experimental design of the rabbit model study. Thirty-six rabbits were used in this study, and the rabbits were randomly divided into six groups, with six rabbits in each group. The rabbits in group A underwent ligature of the vertebral artery via blockage of deep artery flow with simultaneous closure of the common carotid artery valve, thus creating cerebral ischemia by blocking blood flow on both sides of the vertebral and common carotid arteries. For the rabbits in group B, cerebral ischemia was created by the same method as for group A prior to opening of the perfusion tube valve on both sides of the common carotid artery. Protection solution was slowly injected at a speed of 60 drops/min into the brain via both sides of common carotid artery. The experimental method of group C was almost the same as that of group B except that the rabbit brain protection solution was replaced by 5% glucose solution. For group D, the experimental method was similar to that of group B except that the rabbit brain protection fluid was replaced by the rabbit plasma. The experimental methods of group E and group F were the same as those for group B except that the duration of infusion of brain protection solution was increased from 15 min to 30 and 60 min, respectively.
Preparation of the brain, liver, heart, kidney tissue infusions. Six healthy, male or female rabbits (1 year of age and 2 kg) were purchased from Guangxi Animal Center (Nanning, China) and were used for this experiment. The operation was performed via a cervical incision following anesthesia. Fifty milliliters of blood from the carotid artery was added into a beaker containing 0.1 mol/l natrium citricum (1 vol anticoagulant plus 9 vol whole blood). After centrifuging the suspension at 100× g for 10 min, we separated the supernatant. We next took appropriate amounts of the rabbit brain, liver, heart, or kidney tissue and ground them to a powder. One gram of each ground tissue was mixed with 10 ml of saline to make the tissue suspension. Following centrifugation, the supernatant was separated to make the brain infusion, liver infusion, heart infusion, and kidney infusion for later use.
Experimental procedures of blood coagulation measurement. To examine the effect of brain tissue infusion on blood coagulation as compared to the effect of liver infusion, heart infusion, and kidney infusion in vitro, we took five tubes and 0.6 ml citrate plasma from the same rabbit was added to each tube. This was followed by the addition of 0.2 ml brain infusion, liver infusion, heart infusion, and kidney infusion to tube 1, tube 2, tube 3, and tube 4, respectively. As a control, 0.2 ml of normal saline was added to tube 5. The contents of all of the tubes were adequately mixed. Finally, 0.2 ml 1/40 M calcium chloride solution was added to each of the five tubes. Blood clotting time of each tube was recorded. The tubes were shaken once when clotting time and then every 10 s until the experiment was ended. The same protocol was then applied to five additional rabbits. The average blood coagulation time of each tube for the six rabbits was calculated (Table I).
Statistical analysis. Data are presented as mean±SD. SPSS 17.0 (SPSS Inc., Chicago, IL, USA) and Excel (Microsoft, Redmond, WA, USA) software were used for statistical analysis. Data were analyzed by ANOVA and the Student Newman Keuls post-hoc test. p<0.05 was considered to indicate a statistically significant difference.
Results
Effects of different perfusion solutions on recovery from cerebral dormancy in rabbits. To assess the effect of our cerebral protective solution on achieving recovery from cerebral dormancy in an established rabbit model of occlusion-reperfusion (6), we randomly divided 36 rabbits into six groups, with six rabbits in each group.
The results for group A showed the following: cessation of breathing was achieved within 10-15 s following brain ischemia; EEG tracings gradually disappeared within 13 to 18 s; blood pressure increased within 1-2 min and then rapidly dropped to a state of shock. Rabbits were given artificial chest compression rescue breathing and following blockage of blood flow for 7.5 min, all of the cerebral blood flow ceased prior to the rescue. The rabbits were not able to restore spontaneous breathing and died. Observation was carried-out for over 10 min on the head of the dead rabbit, and the brain appearance was characterized by: bulging; meningeal tension; change in surface color to dark red; flattened sulci; narrowing of monoventricular cavity; and brain edema (Table II).
Results for the rabbits in group B showed that breathing and blood pressure in this group remained stable, and EEG gradually disappeared 15-20 s after blocking cerebral blood flow. After 15 min, infusion of the brain protection fluid was stopped and blood flow was restored to both sides of the common carotid and vertebral arteries. Blood pressure and breathing in group B remained stable and electrical activity was gradually restored. When the anesthetic effect disappeared, the rabbits survived and were able to climb up, walk and recover their reflexes. Two hours later, craniotomy observation was carried-out and it was found that the rabbit brain was normal in appearance and had good blood supply (Table II).
Preparation of the blood coagulation experiment.
However, the rabbits in group C were unable to maintain smooth breathing and blood pressure during the process of infusion, except when they were given artificial chest compressions. As in group B, infusion of brain perfusion fluid was stopped at 15 min with subsequent restoration of blood flow through the common carotid and vertebral arteries. The rabbits in group C died due to loss of spontaneous breathing. Craniotomy observation revealed that the appearance of the rabbit brain was the same as that in group A (Table II).
Similarly, rabbits in group D were unable to maintain smooth breathing and blood pressure during the process of infusion except when being given artificial chest compressions. Upon cessation of infusion of brain perfusion fluid at 15 min, rabbits in group D died due to loss of spontaneous breathing. The appearance of the brain upon craniotomy was the same as in group A (Table II).
Interestingly, 5 rabbits of group E had stable blood pressure and breathing, with gradual restoration of electrical activity. Once the anesthetic effect disappeared, the rabbits survived and were able to climb up, walk and recover their reflex. The remaining rabbits of this group died due to loss of spontaneous breathing. Craniotomy was carried-out 2 h after death and it was found that the rabbit brain had a normal appearance and good blood supply (Table II).
Not to our surprise, 3 rabbits in group F had stable blood pressure and breathing, with restored electrical activity. When the anesthetic effect disappeared, rabbits survived and they could climb up, walk and recovered their reflex. The remaining rabbits in this group died due to loss of spontaneous breathing. Two hours following death craniotomy observation was carried out and it was found that the rabbit brain was normal in appearance and had good blood supply (Table II).
Effects of different organ tissue infusions on blood coagulation in vitro. Stroke incidence is high in the clinic and the rate of thrombosis is higher in the brain as compared to other parts of the body. We were interested to understand whether there is something present in the brain tissue that affects or influences blood coagulation and thrombokinesis. To address this question, we assessed the effect of brain tissue infusion on blood coagulation, compared to the effect of liver infusion, heart infusion, and kidney infusion in vitro. As seen in Table III, the blood clotting time of tube 1 (brain infusion) was 1’00”, which was the shortest, while the blood clotting time of tube 5 (normal saline) was 5’02”, which was the longest. The order of the average clotting time among the 5 tubes was: tube 1 (brain infusion) <tube 2 (liver infusion) <tube 4 (kidney infusion) <tube 3 (heart infusion) <tube 5 (normal saline). These data clearly show that the brain infusion led to a statistically significant faster rate of blood coagulation than for any of the other tissue infusions tested with (Table III).
Effect of perfusion of cerebral protective solution on cerebral dormancy recovery in a rabbit model of occlusion-reperfusion.
Discussion
Cerebral ischemic diseases have great influence on human life and life quality, for which reason they are under intensive basic and clinical investigations. Due to the unique structure and function of the brain, it is difficult to study cerebral ischemic diseases in animal models in vivo (7). Following cerebral ischemia, the brain stops functioning, and conventional methods cannot restore its function. This leads to an impression that brain death occurs rapidly following cerebral ischemia. This has led to the belief that the brain is sensitive to ischemia and hypoxia and that irreversible brain death occurs after lack of blood supply for 3 to 5 min.
In the current study, we found that cessation of electrical activity in loss of brain function after a lack of blood supply were only signs of a dormant state of brain cells, but not of true brain death. With the return of blood flow (reperfusion), brain cells were re-awakened and regained their function and biological activities. The key to the restoration of cellular function was the restoration of blood supply, which prevented the occurrence of ischemic brain edema and blood clotting. We demonstrated that the cerebral dormancy period was prolonged up to 60 min when a cerebral protective solution was used after the onset of arterial occlusion and before reperfusion. This notion is supported by our observation that neurons are still active hours after the brain slices are made, as determined by recording of neuronal electrical activity (8), suggesting that the brain has a good capacity to tolerate ischemia and hypoxia. In the clinic, ‘ordinary’ brain-dead patients show such traditional signs of life as warm, moist skin, a palpable pulse, and respiration. It is therefore not surprising that people believe that ‘ordinary brain death’ is a separate entity that occurs prior to ‘real brain death’. In the long term, as more is learned about neurological function and especially as means are developed for the replacement or regeneration of neurological function, the notion of irreversible loss of brain function will likely need to be revisited. The definition of brain death and the standard and criteria for clinical diagnosis of brain death may need to be modified and revised.
Comparison of the effect of different organ tissue infusions on blood clotting time in vitro.
In a separate study, we observed by autopsy that brain edema occurs rapidly at death in the rabbit models of cerebral ischemia and reperfusion, whereas rabbits that died of brain ischemia without reperfusion do not have obvious brain edema (1). This suggests that when the brain lacks a blood supply, brain tissues absorb water quickly. With reperfusion, this rapidly absorbed fluid from the blood causes cerebral edema. As a consequence, the blood in the brain becomes more concentrated and increases in viscosity, leading to an increase in resistance to blood flow. On the other hand, the quickly absorbed fluid increases hydrostatic pressure of the brain tissue and therefore brain perfusion pressure. Due to the fixed size of the cranial cavity, brain edema and increased brain pressure in turn lead to vascular compression and blood stasis, which reduces brain perfusion. In the present study we also showed that blood coagulation was faster for brain infusion than for infusion of any other tissue, including heart, kidney and liver, under equal conditions. In addition, enhanced blood viscosity and blood stasis as a result of water absorbed by the brain tissue speeded-up blood clotting. Taken together, these factors increase blood stasis and blood siltation in a vicious cycle. Above all, the net result is that brain perfusion is inadequate and brain death occurs rapidly. Therefore, brain edema, cerebral blood clotting and thrombosis are the major causes of brain death after cerebral ischemia and reperfusion. However, why the brain tissues absorb water quickly after a lack of blood supply and why the clotting speed is increased in the brain as compared to other organs and tissues of the body are questions that remain to be experimentally determined.
The main component of the cerebral protective solution used in this study is dextrane. The molecular weight of dextrane is similar to that of plasma albumin, meaning that it can quickly increase plasma volume and plasma colloidal osmotic pressure. Dextrane reduces blood viscosity through disaggregation of gathered red blood cells and platelets. Besides its anti-platelet effect, dextrane has anticoagulant properties: it can help restrain the activation of blood coagulation factor II and reduce activity of blood clotting factor I and factor VIII, thus preventing thrombus formation (9). Additionally, the crystal osmotic pressure of the brain protection solution is equal to that of plasma, therefore it does not affect the water exchange into and out of the cell, preventing the development of intracellular edema. Because our brain protection solution has twice the colloidal osmotic pressure of normal plasma, it strongly prevents the formation of brain edema after ischemic anoxia. For these reasons, perfusion of this brain protection solution in our experiments considerably extended cellular dormancy of the rabbits for up to 60 min. In comparison, the rabbits perfused with plasma (normal colloidal pressure, group C) or those treated with 5% glucose solution (no colloidal pressure, group D), all died of cerebral edema and cerebral hemorrhage, respectively. This can be explained because plasma and 5% glucose solution do not exhibit any anticoagulation or anti-water absorption action following cerebral ischemia and anoxia. These observations suggest that the anticoagulation and anti-water absorption properties of our brain protection solution are critical for its prevention of brain edema and prolongation of dormancy in the experimental rabbits.
In 1985, when conducting basic research on brain ischemia-reperfusion injury, the researchers find that free radicals are involved in the process (10). Currently, it has been shown that free radicals, calcium overload, excitatory amino acid, nitric oxide, pro-inflammatory reactions, and apoptosis are all involved in cerebral reperfusion injury (11-17). In the present study, we demonstrated that the causes of cerebral ischemia-reperfusion injury were cerebral edema via rapid water absorption and cerebral blood flow blockage secondary to blood clotting. This notion is also supported by the effectiveness of mannitol, as well as anti-coagulant and anti-platelet drugs (18), in cerebral ischemic disease. However, whether it is feasible to directly inject agents with high colloidal osmotic pressure and anti-clotting activity into the ischemic brain for the recovery of brain function following cerebral infarction remains to be clinically demonstrated. In addition, it is necessary to investigate the mechanism by which brain function, electrical activity, and brainstem functions, such as maintenance of breathing and blood pressure, can be recovered when the brain protection solution is perfused for 30-60 min after the onset of arterial occlusion and before the return of blood flow.
In summary, the present study shows that perfusion of the cerebral protective solution produced a significant neuroprotective benefit of and prolonged cerebral dormancy time in our rabbit model of occlusion-reperfusion. We also found that blood clotting occurred faster in the presence of the brain tissue infusion than the liver, kidney, and heart tissue infusions, indicating the possible higher rate of thrombosis in the brain than in any of the other organs tested. Although further studies are required to elucidate why blood coagulation in the brain may be faster than in the other tissues of the body, as well as the mechanisms underlying this phenomenon, the findings in this study provide a scientific basis for developing a cerebral protective agent as a potentially more effective therapeutic regimen for treatment of patients with stroke and cerebral ischemic diseases.
Acknowledgements
This study was supported by a grant from the Guangxi University of Chinese Medicine (No. P2010092).
- Received April 27, 2014.
- Revision received June 1, 2014.
- Accepted June 2, 2014.
- Copyright © 2014 The Author(s). Published by the International Institute of Anticancer Research.
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