- Email: [email protected]
A new model for total cerebral ischemia in dogs
Resuscitation, 13 (1986) 233-242 Elsevier Scientific Publishers Ireland
233 Ltd.
A NEW MODEL FOR TOTAL CEREBRAL
TATSURU ARAI, HITOSHI IMON
ISA0
TSUKAHARA,
Department of Anesthesiology, Ehime Onsen-gun, Ehime-ken, 791-02 (Japan) (Received (Revision (Accepted
March lst, 1986) received May 19th, May 21st, 1986)
ISCHEMIA
KENTARO
DOTE,
University School
IN DOGS
KOH
of Medicine,
KUZUME
and
Shigenobu-cho,
1986)
SUMMARY
We have developed a new method producing total cerebral ischemia (TCI) in dogs; clamping ascending aorta with aorto-atrial bypass formation. Clamping ascending aorta provides TCI, the duration of which can be controlled up to the periods of 10 min. Beyond this interval, it is difficult to maintain TCI because of heart failure from high afterload. Blood outflow from left ventricle is completely obstructed except for coronary circulation which is small relative to the blood volume expelled from left ventricle, even if venous return to the heart is reduced. Aorto-atrial bypass formation during aortic clamping provides two distinctive advantages. First, adjusting aortic pressure in an appropriate level low enough not to overload myocardium but still high enough to maintain sufficient coronary blood flow is possible by regulating the blood flow through the bypass tubing, and secondly drug administration and blood volume control is possible through the tubing. These result in better preservation of myocardium, enabling longer TCI and longer survivals after TCI. We were successful in having up to 18 min of TCI with this method. Seventy-five percent of dogs of 12 min TCI and 40% of 15 min TCI survived 7 days, limit of experiment, after TCI, but no dogs of 18 min TCI survived for more than 3 days. Key words: Total cerebral atria1 bypass formation
ischemia
model
-
Aortic
clamping
-
Aorto-
INTRODUCTION
In order to determine the relationship between the duration of TCI and the severity of ischemic-anoxic encephalopathy, or other specific neuro0300-9572/86/$03.50 Printed and Published
o 1986 in Ireland
Elsevier
Scientific
Publishers
Ireland
Ltd.
234
logical deficits, it is necessary to have specific animal models in which the following are satisfied: (1) TCI can be kept long enough to meet the purpose of the particular experiment; (2) controllable restoration of cerebral perfusion to normal pressure is possible at the end of the TCI period; (3) the circulatory status thereafter should be stable enough to allow for short-term or longterm survivals. We have developed a new TCI method which satisfies all the above criteria fairly well, i.e. clamping ascending aorta with aorto-atrial bypass formation in dogs. With this model there has been little difficulty in having a TCI of 15-18 min, a length which can cover various types of TCI experiments. The method needs rather complicated surgery but the reliability in obtaining long term TCI and survival outstrips this drawback. METHODS
Thirty-one adult mongrel dogs weighing B- 14 kg were anesthetized with thiopental (10 mg/kg). Following tracheal intubation the animals were ventilated with volume cycle respirator (Acoma Animal). The PO2 was kept between 100-150 mmHg and PCO* 34-40 mmHg. Body temperature was maintained at 37-38°C monitoring esophageal temperature. Anesthesia was maintained with 0.5-1.0% halothane during preparatory surgery, and pancuronium bromide was given for muscle relaxation. MAP (left femoral artery), EKG (bipolar standard leads) and EEG (bipolar parietal and occipital leads) were recorded continuously on a 6 channel strip chart recorder. The dogs were divided into 4 groups according to the length of the ischemia desired; 9 min (n = 5), 12 min (n = 8), 15 min (n = 12) and 18 min (n = 6). The animals were placed in left lateral position and the right thoracic skin was shaved precautionarily. All surgical procedures were done by aseptic technique. A right-side thoracotomy was made in the 4th intercostal space. Umbilical tapes were placed around superior vena cava (SVC), inferior vena cava (IVC), azygous vein and ascending aorta. Around the aorta two tapes were placed to ease surgical manipulation of it. The bypass tubing was hand-made. Atria1 end of the tubing, filled with heparinized saline, was inserted into right auricle following an incision on it and secured by tying. After lowering systolic pressure to 80-100 torr by partial ligation of IVC, aortic tip of tubing was inserted into ascending aorta through a small incision and fixed tightly using mattress suture with felt (Fig. 1). Clamping of the vessels was done in the sequence of IVC, ascending aorta and SVC in order to avoid overfilling of the heart and congestion of the brain. Azygous vein was usually left open, but if heart filling was more than sufficient it was closed. As soon as aorta was clamped the bypass tubing was opened. Bypass blood flow was regulated with finger pressure applied to the tubing in order to maintain mean aortic pressure (measured in the bypass tubing) 60-90 torr or systolic 100-150 torr. Aortic pressure became higher when the tubing was squeezed strongly and vice versa. If it was necessary to increase blood volume in the heart to maintain appropriate aortic pressure,
235
Azygous V.
i
Fig. 1. Schematic for the aorto-atria1 bypass formation. Aortic tip of the bypass tubing was made from an Aortic Arch Cannula (3.8 mm) from Sarns Inc.
the ligation of IVC was released transiently, and to decrease, blood was aspirated through the tubing. A small dose of epinephrine (less than l/40 mg) was given through the tubing to support the cardiac contraction during aortic clamping and peri-declamping period, if necessary. During aortic clamping the dogs received 100% oxygen but ventilator-y patterns were not changed from that of preclamping period. At the end of TCI period the ligation of SVC was released first, then the aortic clamp and all other ligations together, immediately followed by closing of bypass tubing. NaHCO, (40--50 mEq), and if necessary, a small dose of epinephrine were administered into right atrium through the bypass tubing. During this period animals were hyperventilated several times manually to inflate the lungs fully. After the circulatory status was stabilized, tubing was removed. The incision in the aortic wall was closed tightly with the mattress suture which had secured the bypass tubing on the aorta. Thoracotomy was closed after careful checking for bleeding. Controlled ventilation, with 100% oxygen for the first 2-3 h and later with 30-50%, was continued at least 24 h in the TCIs beyond 12 min to maintain PO, above 120 mmHg and BCO, 30-40 mmHg. Microdoses of dopamine were administered to maintain systolic pressure above 120 torr during postischemic period, if necessary. All dogs received antibiotics everyday during post-ischemic period. Dogs were observed for 7 days (or until maintaining of normal arterial pressure became difficult) for the recovery of neurological function.
236
The totality of cerebral ischemia with this method was evaluated in 2 Center, Amersham) bovine serum albumin dogs. “’ I-labelled (Radiochemical was injected into the right atrium through the bypass tubing immediately after aortic clamping, and the radioactivity of the blood in femoral artery, superior saggital sinus, and bypass tubing was measured with Auto Well Gamma System (Aloka, Tokyo). The lengths of ischemias were 15 min. Blood sampling was done before clamping, 5 min and 15 min after the start of clamping, and 5 min after the declamping. RESULTS
Following complete ligation of IVC arterial pressure. decreased acutely within seconds (Fig. 2), resulting in softened aorta which enabled us to clamp it easier. EEG became isoelectric within 25 s after aortic clamping. All the dogs had fixed and dilated pupils within 40 s. Immediately following aortic clamping heart contraction became vigorous and increased in rate for 2-4 min. During this period supraventricular or ventricular dysrythmias and/or bradyarythmias were observed in most dogs. Lidocaine (2-5 mg) was given to 11 dogs for ventricular dysrythmias, and 0.1-0.05 mg atropine to 3 dogs for bradyarythmias. Usually after 2-4 min contraction slowed and stabilized, and after 7-10 min it became weaker over time. Administration of epinephrine was needed in 1 dog before 12 min. It was given to 3 dogs of 15 min and 2 dogs of 18 min TCI near the end of the TCI period in order to augment the weakened heart contraction, and more importantly to overcome the declamping shock due to the returning of strongly acidic venous blood to the heart immediately after the release of ligations of great vessels. Blood gases, blood glucose and serum electrolytes measured in the bypass
FEMORAL PRESSURE
BYPASS PRESSURE
EEG
Fig. 2. Changes in arterial pressure, bypass tubing pressure and EEG during clamping and declamping period of ascending aorta in a dog of 15 min TCI.
*Significantly different a - = no data.
4.3kO.4 8
0.06 7.5 2.3 1.9
K’ N
10
? t ? +
124 ? 32 14223
2
7.43 126 36.6 m-2.3
Glucose Na+
N
PH PO* PC0 BE
of studies.
BLOOD
Before clamping
GAS.
BLOOD
N = number
I
TABLE
AND
SERUM
ELECTROLYTES
0.09* 45 5.2* 2.4
from pre-clamping
-
-” -
10
t + ? +
value (P 5 0.05).
-
-
10
7.58 ? O.lO* 3412 49 20.6 2 4.5* -0.9 +_ 2.5
3
1
7.51 330 25.0 -1.3
(min)
During clamping
10
t + + +
0.09* 52 5.3* 2.9
3.8 + 0.4 8
96 f 18 143 !: 3
7.57 346 19.3 -1.1
6
10
? + f. t
O.lO* 52 5.6* 3.4
4.0 2 0.3 8
108 t 33 145 26
7.59 361 17.6 -1.6
9
8
? k + ? 0.16* 80 5.5* 4.2
TUBING
3.9 + 0.4 8
90 + 21* 144 ? 7
7.64 341 16.1 -0.8
12
0,.
IN THE BYPASS
are under 100%
MEASURED
Values are mean k S.D. PO, values during clamping
GLUCOSE
8
? + ? f
0.23* 119 6.8* 6.5
3.820.6 8
77f18* 147 26
7.65 329 15.6 0.8
15
-
-
7.66 335 16.0 -0.3
18
4
+ + ? +
0.19 121 7.9* 4.7
238
tubing during aortic clamping are shown in Table I. PCOz decreased rapidly resulting in rapid increase in pH during early period of clamping. The rate of pH increase slowed after 3 min. Blood glucose decreased significantly after 12 min, but changes in Na+ and K’ values were not significant for the periods of 15 min. MAP immediately after the release of aortic clamping was 50-100 torr. It usually increased to above 150 torr within 1 min and remained there for several minutes, though these values were affected considerably by the administration of the catecholamines during peri-declamping period (Fig. 2). Following this transient increase it decreased gradually. During this decrease lo-20 min of dopamine administration was needed in 3 dogs of 15 min and 4 dogs of 18 min TCI. Except for early period of aortic clamping dysrythmias were rarely seen throughout entire experiment. Also, except for depression of ST no signs of myocardial infarction on EKG were observed in any dogs. We lost 2 dogs during aortic clamping due to contraction failure followed
=
18 min
m
Contro” ed ventilation
w
Spontaneour
_
Complete Recovery
respiration
_
Wand,
Eat etc.)
G
kl
.. ,.,.:
A..
-I
12
:.:.....,
..
:,.:A.....:.:..
:~::.::..:::.:, y.:.: .,... :.:.:.>8:’
min
, t __I I LI
,
I
1
2
3
4
5
6
7
(days)
Fig. 3. Relationship between the length of TCI and recovery of neurologic functions after TCI. One bar represents one dog.
239
by ventricular ‘fibrillation (1 each in 15 min and 18 min TCI), 1 dog (15 min TCI) due to declamping shock and 1 dog due to mechanical failure of the respirator during post-operative course. The length of survival and degree of neurologic function after respective lengths of TCI of remaining 27 dogs are shown roughly in Fig. 3. Precise timing of reappearance of EEG was difficult to discern, but it was usually observed in all groups 2-3 h after the start of recirculation. Seventy-five percent of dogs of 9 and 12 min TCI and 40% of 15 min TCI survived 7 days after TCI, limit of experiment, but after 18 min of TCI no dogs could survive for more than 3 days and all dogs remained comatose with no spontaneous respiration until they died. Levels of radioactivity in the bypass tubing and peripheral blood during 15 min aortic clamping in additional 2 dogs are shown in Table II. The background count was around 80/min. The radioactivity of the peripheral blood did not change and actually kept the same background order during 15 min of aortic clamping in spite of 700-25 000 times higher radioactivity in the bypass tubing. These results show there was no leakage of blood through aortic clamping during 15 min of ischemia. DISCUSSION
So far various kinds of TCI methods have been developed in dogs, total isolation of cerebral circulation (Kabat and Dennis, 1938; White and Donald, TABLE II MEASUREMENT
OF RADIOACTIVITY
Abbreviations: F,, femoral artery; S,, superior saggital sinus; Bt, bypass tubing. Numbers in column 2: 1, preclamping; 2,5 min after beginning of clamping; 3, 15 min after beginning of clamping; 4, post-declamping. Countlmin Dog 1 1
F,
2 3 4 1
S,
2 3 4 1
Bt
2 3 4
81 72 82 8491
Dog 2 78 77 87 19 432
87 74 132 8894
76 98 17 267
83 64 784 67 054 39 205
74 196 285 196 759 57 815
80
210
1962; Lind, Snyder, Kampschulte and Safar, 1975), ventricular fibrillation (Safar, Stezoski and Nemoto, 1976), ligation of’various arteries (Lind et al., 1975), aortic clamping (Brockman and Jude, 1960; Lind et al., 1975; Matsumae, Iijima and Yonezawa, 1984), etc. But none of them satisfactorily covers all the criteria for TCI model we mentioned above. Among them clamping of ascending aorta has distinctive advantages: technical simplicity and preciseness of timing in initiating and terminating TCI, though it needs thoracotomy and preparation of aorta. One of the weak points of this method is early exhaustion of myocardium because of high afterload due to highly elevated aortic pressure resultant from the obstruction of blood outflow from the heart except for coronary artery. Therefore it is necessary to decrease aortic pressure in this method. We had been trying to decrease aortic pressure by reducing venous return to the heart through ligating IVC and SVC, and also occasionally azygous vein during aortic clamping. But it was still not easy to have TCI longer than 10 min. Though myocardium exhibited vigorous contraction at first, it exhausted itself gradually after several minutes. We assumed it was due to still high afterload. Even though venous return to the heart was arrested the blood volume expelled from left ventricle was disproportionately large relative to the diameter of coronary artery. Mean aortic pressure following aortic clamping usually exceeded 200 torr with this method. If clamping of the aorta is delayed for more than 10 s after the arrest of venous return, the blood volume in the heart and pulmonary vasculature is greatly decreased, resulting in decrease in sonic pressure during aortic clamping. We had tried this technique too, but to maintain appropriate coronary perfusion pressure was not without problems and the results were poor. Our new method aimed to regulate aortic pressure at an appropriate level low enough not to overload myocardium but still high enough to maintain sufficient coronary blood flow. Venous return to the heart was reduced first, and then finer control of aortic pressure was made possible by regulating blood flow through the bypass tubing. In our study mean aortic pressure of 60-90 torr brought about the best results. An additional advantage with this method was that administration of drugs and/or regulation of the blood volume in the heart and lungs were possible through the tubing during aortic clamping. These provided better preservation of myocardium and increased the safety margin greatly. Lind et al. (1975) reported they were successful having 15 min of TCI in 5 dogs with ordinary aortic clamping method, reducing venous return by ligating IVC and SVC. But no dogs survived more than 20 h after the aortic clamping due to severe neurological sequelae, and several attempts to extend the experiments or to obtain long-term survival were discouraged by post-thoracotomy complications, difficulty in maintaining life support, etc. With our new method circulatory status was fairly stable after 15 min of aortic clamping and more than 50% of the dogs survived for 7 days. Also we could obtain 18 min of TCI, after which dogs could survive 2-3 days. The difference in results between ours and theirs appears to be due to
241
the better preservation of myocardium in our animals during aortic clamping; because of it myocardium could tolerate longer periods of TCI and dogs could survive longer after the TCI. One of the drawbacks of our method is that it requires complicated surgery. As a matter of fact we had to discontinue the experiment in 3-4 dogs during preliminary study due to hemorrhages, mostly at puncture site of aorta. But after these setbacks, with the improvement of method and surgical technique we have been having little trouble related to the surgical procedures. Jackson and Dole (1979) were successful in having 12 min of TCI by occluding ascending aorta with a balloon introduced via femoral artery. This method does not need thoracotomy and provides fairly long TCI, but the effect on myocardium is practically the same as ordinary aortic clamping method. In the animals which could survive for 5-7 days no recovery of neurologic function was noted and all remained deeply comatose. In our experiment 60% dogs could stand by the 3rd day after 12 min of TCI. We assume the difference between ours and theirs is again due to better preservation of myocardium in our method, resulting in better perfusion to the brain during post-ischemic period facilitating the recovery of brain function. We believe that the reliability in obtaining long term TCI outstrips the drawbacks related to the complicated surgery in our method. In this study we did not change pre-clamping ventilatory pattern during aortic clamping. Therefore the increase in pH at the end of each TCI period was considerable. At the beginning of this experiment we tried to normalize blood gases by changing ventillation volume. Decreasing ventilation volume occasionally resulted in a disproportionate increase in PCO, and decrease in pH. Normalization of blood gas values by adding CO, or adjusting ventilation volume may result in better outcome than ours. There were no significant changes in Na’ and K’ levels during aortic clamping but blood glucose decreased significantly. Adding glucose through the tubing may be beneficial. We have not tried TCI longer than 18 min. The reason is that for the evaluation of neurological deficit due to cerebral ischemia, we consider it unnecessary to have longer TCI than 18 min, since no dogs that experienced this length of TCI showed any recovery of cerebral function except for EEG; all remained deeply comatose until they died. We assume that 15 min is the critical length of ischemia for the reversibility and irreversibility of cerebral function in dogs. REFERENCES Brockman, S.K. and Jude, J.R. (1960) The tolerance of the dog brain to total arrest of circulation. Bull. Johns Hopkins Hosp., 106, 74-80. Jackson, D.L. and Dole, W.P. (1979) Total cerebral ischemia: a new model system for the study of post-cardiac arrest brain damage. Stroke, 10, 38-43. Kabat, H. and Dennis, C. (1938) Decerebration in the dog by complete temporary anemia of the brain. Proc. Sot. Biol. Med., 38, 864-865.
242 Lind, B., Snyder, J., Kampschulte, S. and Safar, P. (1975) A review of total brain ischemia models in dogs and original experiments on clamping the aorta, Resuscitation, 4, 1931. Matsumae, T., Iijima, K. and Yonezawa, T. (1984) Experimental model producing global brain ischemia by clamping the aorta in dogs. Brain Nerve (Tokyo), 36, 349-355. Safar, P., Stexoski, W., Nemoto, E.M. (1976) Amelioration of brain damage after 12 minutes cardiac arrest in dogs. Arch. Neurol., 33, 91-95. White, R.J. and Donald, D.E. (1962) Basilar artery ligation and cerebral ischemia in dogs. Arch. Surg., 84,470-475.
233 Ltd.
A NEW MODEL FOR TOTAL CEREBRAL
TATSURU ARAI, HITOSHI IMON
ISA0
TSUKAHARA,
Department of Anesthesiology, Ehime Onsen-gun, Ehime-ken, 791-02 (Japan) (Received (Revision (Accepted
March lst, 1986) received May 19th, May 21st, 1986)
ISCHEMIA
KENTARO
DOTE,
University School
IN DOGS
KOH
of Medicine,
KUZUME
and
Shigenobu-cho,
1986)
SUMMARY
We have developed a new method producing total cerebral ischemia (TCI) in dogs; clamping ascending aorta with aorto-atrial bypass formation. Clamping ascending aorta provides TCI, the duration of which can be controlled up to the periods of 10 min. Beyond this interval, it is difficult to maintain TCI because of heart failure from high afterload. Blood outflow from left ventricle is completely obstructed except for coronary circulation which is small relative to the blood volume expelled from left ventricle, even if venous return to the heart is reduced. Aorto-atrial bypass formation during aortic clamping provides two distinctive advantages. First, adjusting aortic pressure in an appropriate level low enough not to overload myocardium but still high enough to maintain sufficient coronary blood flow is possible by regulating the blood flow through the bypass tubing, and secondly drug administration and blood volume control is possible through the tubing. These result in better preservation of myocardium, enabling longer TCI and longer survivals after TCI. We were successful in having up to 18 min of TCI with this method. Seventy-five percent of dogs of 12 min TCI and 40% of 15 min TCI survived 7 days, limit of experiment, after TCI, but no dogs of 18 min TCI survived for more than 3 days. Key words: Total cerebral atria1 bypass formation
ischemia
model
-
Aortic
clamping
-
Aorto-
INTRODUCTION
In order to determine the relationship between the duration of TCI and the severity of ischemic-anoxic encephalopathy, or other specific neuro0300-9572/86/$03.50 Printed and Published
o 1986 in Ireland
Elsevier
Scientific
Publishers
Ireland
Ltd.
234
logical deficits, it is necessary to have specific animal models in which the following are satisfied: (1) TCI can be kept long enough to meet the purpose of the particular experiment; (2) controllable restoration of cerebral perfusion to normal pressure is possible at the end of the TCI period; (3) the circulatory status thereafter should be stable enough to allow for short-term or longterm survivals. We have developed a new TCI method which satisfies all the above criteria fairly well, i.e. clamping ascending aorta with aorto-atrial bypass formation in dogs. With this model there has been little difficulty in having a TCI of 15-18 min, a length which can cover various types of TCI experiments. The method needs rather complicated surgery but the reliability in obtaining long term TCI and survival outstrips this drawback. METHODS
Thirty-one adult mongrel dogs weighing B- 14 kg were anesthetized with thiopental (10 mg/kg). Following tracheal intubation the animals were ventilated with volume cycle respirator (Acoma Animal). The PO2 was kept between 100-150 mmHg and PCO* 34-40 mmHg. Body temperature was maintained at 37-38°C monitoring esophageal temperature. Anesthesia was maintained with 0.5-1.0% halothane during preparatory surgery, and pancuronium bromide was given for muscle relaxation. MAP (left femoral artery), EKG (bipolar standard leads) and EEG (bipolar parietal and occipital leads) were recorded continuously on a 6 channel strip chart recorder. The dogs were divided into 4 groups according to the length of the ischemia desired; 9 min (n = 5), 12 min (n = 8), 15 min (n = 12) and 18 min (n = 6). The animals were placed in left lateral position and the right thoracic skin was shaved precautionarily. All surgical procedures were done by aseptic technique. A right-side thoracotomy was made in the 4th intercostal space. Umbilical tapes were placed around superior vena cava (SVC), inferior vena cava (IVC), azygous vein and ascending aorta. Around the aorta two tapes were placed to ease surgical manipulation of it. The bypass tubing was hand-made. Atria1 end of the tubing, filled with heparinized saline, was inserted into right auricle following an incision on it and secured by tying. After lowering systolic pressure to 80-100 torr by partial ligation of IVC, aortic tip of tubing was inserted into ascending aorta through a small incision and fixed tightly using mattress suture with felt (Fig. 1). Clamping of the vessels was done in the sequence of IVC, ascending aorta and SVC in order to avoid overfilling of the heart and congestion of the brain. Azygous vein was usually left open, but if heart filling was more than sufficient it was closed. As soon as aorta was clamped the bypass tubing was opened. Bypass blood flow was regulated with finger pressure applied to the tubing in order to maintain mean aortic pressure (measured in the bypass tubing) 60-90 torr or systolic 100-150 torr. Aortic pressure became higher when the tubing was squeezed strongly and vice versa. If it was necessary to increase blood volume in the heart to maintain appropriate aortic pressure,
235
Azygous V.
i
Fig. 1. Schematic for the aorto-atria1 bypass formation. Aortic tip of the bypass tubing was made from an Aortic Arch Cannula (3.8 mm) from Sarns Inc.
the ligation of IVC was released transiently, and to decrease, blood was aspirated through the tubing. A small dose of epinephrine (less than l/40 mg) was given through the tubing to support the cardiac contraction during aortic clamping and peri-declamping period, if necessary. During aortic clamping the dogs received 100% oxygen but ventilator-y patterns were not changed from that of preclamping period. At the end of TCI period the ligation of SVC was released first, then the aortic clamp and all other ligations together, immediately followed by closing of bypass tubing. NaHCO, (40--50 mEq), and if necessary, a small dose of epinephrine were administered into right atrium through the bypass tubing. During this period animals were hyperventilated several times manually to inflate the lungs fully. After the circulatory status was stabilized, tubing was removed. The incision in the aortic wall was closed tightly with the mattress suture which had secured the bypass tubing on the aorta. Thoracotomy was closed after careful checking for bleeding. Controlled ventilation, with 100% oxygen for the first 2-3 h and later with 30-50%, was continued at least 24 h in the TCIs beyond 12 min to maintain PO, above 120 mmHg and BCO, 30-40 mmHg. Microdoses of dopamine were administered to maintain systolic pressure above 120 torr during postischemic period, if necessary. All dogs received antibiotics everyday during post-ischemic period. Dogs were observed for 7 days (or until maintaining of normal arterial pressure became difficult) for the recovery of neurological function.
236
The totality of cerebral ischemia with this method was evaluated in 2 Center, Amersham) bovine serum albumin dogs. “’ I-labelled (Radiochemical was injected into the right atrium through the bypass tubing immediately after aortic clamping, and the radioactivity of the blood in femoral artery, superior saggital sinus, and bypass tubing was measured with Auto Well Gamma System (Aloka, Tokyo). The lengths of ischemias were 15 min. Blood sampling was done before clamping, 5 min and 15 min after the start of clamping, and 5 min after the declamping. RESULTS
Following complete ligation of IVC arterial pressure. decreased acutely within seconds (Fig. 2), resulting in softened aorta which enabled us to clamp it easier. EEG became isoelectric within 25 s after aortic clamping. All the dogs had fixed and dilated pupils within 40 s. Immediately following aortic clamping heart contraction became vigorous and increased in rate for 2-4 min. During this period supraventricular or ventricular dysrythmias and/or bradyarythmias were observed in most dogs. Lidocaine (2-5 mg) was given to 11 dogs for ventricular dysrythmias, and 0.1-0.05 mg atropine to 3 dogs for bradyarythmias. Usually after 2-4 min contraction slowed and stabilized, and after 7-10 min it became weaker over time. Administration of epinephrine was needed in 1 dog before 12 min. It was given to 3 dogs of 15 min and 2 dogs of 18 min TCI near the end of the TCI period in order to augment the weakened heart contraction, and more importantly to overcome the declamping shock due to the returning of strongly acidic venous blood to the heart immediately after the release of ligations of great vessels. Blood gases, blood glucose and serum electrolytes measured in the bypass
FEMORAL PRESSURE
BYPASS PRESSURE
EEG
Fig. 2. Changes in arterial pressure, bypass tubing pressure and EEG during clamping and declamping period of ascending aorta in a dog of 15 min TCI.
*Significantly different a - = no data.
4.3kO.4 8
0.06 7.5 2.3 1.9
K’ N
10
? t ? +
124 ? 32 14223
2
7.43 126 36.6 m-2.3
Glucose Na+
N
PH PO* PC0 BE
of studies.
BLOOD
Before clamping
GAS.
BLOOD
N = number
I
TABLE
AND
SERUM
ELECTROLYTES
0.09* 45 5.2* 2.4
from pre-clamping
-
-” -
10
t + ? +
value (P 5 0.05).
-
-
10
7.58 ? O.lO* 3412 49 20.6 2 4.5* -0.9 +_ 2.5
3
1
7.51 330 25.0 -1.3
(min)
During clamping
10
t + + +
0.09* 52 5.3* 2.9
3.8 + 0.4 8
96 f 18 143 !: 3
7.57 346 19.3 -1.1
6
10
? + f. t
O.lO* 52 5.6* 3.4
4.0 2 0.3 8
108 t 33 145 26
7.59 361 17.6 -1.6
9
8
? k + ? 0.16* 80 5.5* 4.2
TUBING
3.9 + 0.4 8
90 + 21* 144 ? 7
7.64 341 16.1 -0.8
12
0,.
IN THE BYPASS
are under 100%
MEASURED
Values are mean k S.D. PO, values during clamping
GLUCOSE
8
? + ? f
0.23* 119 6.8* 6.5
3.820.6 8
77f18* 147 26
7.65 329 15.6 0.8
15
-
-
7.66 335 16.0 -0.3
18
4
+ + ? +
0.19 121 7.9* 4.7
238
tubing during aortic clamping are shown in Table I. PCOz decreased rapidly resulting in rapid increase in pH during early period of clamping. The rate of pH increase slowed after 3 min. Blood glucose decreased significantly after 12 min, but changes in Na+ and K’ values were not significant for the periods of 15 min. MAP immediately after the release of aortic clamping was 50-100 torr. It usually increased to above 150 torr within 1 min and remained there for several minutes, though these values were affected considerably by the administration of the catecholamines during peri-declamping period (Fig. 2). Following this transient increase it decreased gradually. During this decrease lo-20 min of dopamine administration was needed in 3 dogs of 15 min and 4 dogs of 18 min TCI. Except for early period of aortic clamping dysrythmias were rarely seen throughout entire experiment. Also, except for depression of ST no signs of myocardial infarction on EKG were observed in any dogs. We lost 2 dogs during aortic clamping due to contraction failure followed
=
18 min
m
Contro” ed ventilation
w
Spontaneour
_
Complete Recovery
respiration
_
Wand,
Eat etc.)
G
kl
.. ,.,.:
A..
-I
12
:.:.....,
..
:,.:A.....:.:..
:~::.::..:::.:, y.:.: .,... :.:.:.>8:’
min
, t __I I LI
,
I
1
2
3
4
5
6
7
(days)
Fig. 3. Relationship between the length of TCI and recovery of neurologic functions after TCI. One bar represents one dog.
239
by ventricular ‘fibrillation (1 each in 15 min and 18 min TCI), 1 dog (15 min TCI) due to declamping shock and 1 dog due to mechanical failure of the respirator during post-operative course. The length of survival and degree of neurologic function after respective lengths of TCI of remaining 27 dogs are shown roughly in Fig. 3. Precise timing of reappearance of EEG was difficult to discern, but it was usually observed in all groups 2-3 h after the start of recirculation. Seventy-five percent of dogs of 9 and 12 min TCI and 40% of 15 min TCI survived 7 days after TCI, limit of experiment, but after 18 min of TCI no dogs could survive for more than 3 days and all dogs remained comatose with no spontaneous respiration until they died. Levels of radioactivity in the bypass tubing and peripheral blood during 15 min aortic clamping in additional 2 dogs are shown in Table II. The background count was around 80/min. The radioactivity of the peripheral blood did not change and actually kept the same background order during 15 min of aortic clamping in spite of 700-25 000 times higher radioactivity in the bypass tubing. These results show there was no leakage of blood through aortic clamping during 15 min of ischemia. DISCUSSION
So far various kinds of TCI methods have been developed in dogs, total isolation of cerebral circulation (Kabat and Dennis, 1938; White and Donald, TABLE II MEASUREMENT
OF RADIOACTIVITY
Abbreviations: F,, femoral artery; S,, superior saggital sinus; Bt, bypass tubing. Numbers in column 2: 1, preclamping; 2,5 min after beginning of clamping; 3, 15 min after beginning of clamping; 4, post-declamping. Countlmin Dog 1 1
F,
2 3 4 1
S,
2 3 4 1
Bt
2 3 4
81 72 82 8491
Dog 2 78 77 87 19 432
87 74 132 8894
76 98 17 267
83 64 784 67 054 39 205
74 196 285 196 759 57 815
80
210
1962; Lind, Snyder, Kampschulte and Safar, 1975), ventricular fibrillation (Safar, Stezoski and Nemoto, 1976), ligation of’various arteries (Lind et al., 1975), aortic clamping (Brockman and Jude, 1960; Lind et al., 1975; Matsumae, Iijima and Yonezawa, 1984), etc. But none of them satisfactorily covers all the criteria for TCI model we mentioned above. Among them clamping of ascending aorta has distinctive advantages: technical simplicity and preciseness of timing in initiating and terminating TCI, though it needs thoracotomy and preparation of aorta. One of the weak points of this method is early exhaustion of myocardium because of high afterload due to highly elevated aortic pressure resultant from the obstruction of blood outflow from the heart except for coronary artery. Therefore it is necessary to decrease aortic pressure in this method. We had been trying to decrease aortic pressure by reducing venous return to the heart through ligating IVC and SVC, and also occasionally azygous vein during aortic clamping. But it was still not easy to have TCI longer than 10 min. Though myocardium exhibited vigorous contraction at first, it exhausted itself gradually after several minutes. We assumed it was due to still high afterload. Even though venous return to the heart was arrested the blood volume expelled from left ventricle was disproportionately large relative to the diameter of coronary artery. Mean aortic pressure following aortic clamping usually exceeded 200 torr with this method. If clamping of the aorta is delayed for more than 10 s after the arrest of venous return, the blood volume in the heart and pulmonary vasculature is greatly decreased, resulting in decrease in sonic pressure during aortic clamping. We had tried this technique too, but to maintain appropriate coronary perfusion pressure was not without problems and the results were poor. Our new method aimed to regulate aortic pressure at an appropriate level low enough not to overload myocardium but still high enough to maintain sufficient coronary blood flow. Venous return to the heart was reduced first, and then finer control of aortic pressure was made possible by regulating blood flow through the bypass tubing. In our study mean aortic pressure of 60-90 torr brought about the best results. An additional advantage with this method was that administration of drugs and/or regulation of the blood volume in the heart and lungs were possible through the tubing during aortic clamping. These provided better preservation of myocardium and increased the safety margin greatly. Lind et al. (1975) reported they were successful having 15 min of TCI in 5 dogs with ordinary aortic clamping method, reducing venous return by ligating IVC and SVC. But no dogs survived more than 20 h after the aortic clamping due to severe neurological sequelae, and several attempts to extend the experiments or to obtain long-term survival were discouraged by post-thoracotomy complications, difficulty in maintaining life support, etc. With our new method circulatory status was fairly stable after 15 min of aortic clamping and more than 50% of the dogs survived for 7 days. Also we could obtain 18 min of TCI, after which dogs could survive 2-3 days. The difference in results between ours and theirs appears to be due to
241
the better preservation of myocardium in our animals during aortic clamping; because of it myocardium could tolerate longer periods of TCI and dogs could survive longer after the TCI. One of the drawbacks of our method is that it requires complicated surgery. As a matter of fact we had to discontinue the experiment in 3-4 dogs during preliminary study due to hemorrhages, mostly at puncture site of aorta. But after these setbacks, with the improvement of method and surgical technique we have been having little trouble related to the surgical procedures. Jackson and Dole (1979) were successful in having 12 min of TCI by occluding ascending aorta with a balloon introduced via femoral artery. This method does not need thoracotomy and provides fairly long TCI, but the effect on myocardium is practically the same as ordinary aortic clamping method. In the animals which could survive for 5-7 days no recovery of neurologic function was noted and all remained deeply comatose. In our experiment 60% dogs could stand by the 3rd day after 12 min of TCI. We assume the difference between ours and theirs is again due to better preservation of myocardium in our method, resulting in better perfusion to the brain during post-ischemic period facilitating the recovery of brain function. We believe that the reliability in obtaining long term TCI outstrips the drawbacks related to the complicated surgery in our method. In this study we did not change pre-clamping ventilatory pattern during aortic clamping. Therefore the increase in pH at the end of each TCI period was considerable. At the beginning of this experiment we tried to normalize blood gases by changing ventillation volume. Decreasing ventilation volume occasionally resulted in a disproportionate increase in PCO, and decrease in pH. Normalization of blood gas values by adding CO, or adjusting ventilation volume may result in better outcome than ours. There were no significant changes in Na’ and K’ levels during aortic clamping but blood glucose decreased significantly. Adding glucose through the tubing may be beneficial. We have not tried TCI longer than 18 min. The reason is that for the evaluation of neurological deficit due to cerebral ischemia, we consider it unnecessary to have longer TCI than 18 min, since no dogs that experienced this length of TCI showed any recovery of cerebral function except for EEG; all remained deeply comatose until they died. We assume that 15 min is the critical length of ischemia for the reversibility and irreversibility of cerebral function in dogs. REFERENCES Brockman, S.K. and Jude, J.R. (1960) The tolerance of the dog brain to total arrest of circulation. Bull. Johns Hopkins Hosp., 106, 74-80. Jackson, D.L. and Dole, W.P. (1979) Total cerebral ischemia: a new model system for the study of post-cardiac arrest brain damage. Stroke, 10, 38-43. Kabat, H. and Dennis, C. (1938) Decerebration in the dog by complete temporary anemia of the brain. Proc. Sot. Biol. Med., 38, 864-865.
242 Lind, B., Snyder, J., Kampschulte, S. and Safar, P. (1975) A review of total brain ischemia models in dogs and original experiments on clamping the aorta, Resuscitation, 4, 1931. Matsumae, T., Iijima, K. and Yonezawa, T. (1984) Experimental model producing global brain ischemia by clamping the aorta in dogs. Brain Nerve (Tokyo), 36, 349-355. Safar, P., Stexoski, W., Nemoto, E.M. (1976) Amelioration of brain damage after 12 minutes cardiac arrest in dogs. Arch. Neurol., 33, 91-95. White, R.J. and Donald, D.E. (1962) Basilar artery ligation and cerebral ischemia in dogs. Arch. Surg., 84,470-475.