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Effect of spinal cord preconditioning on paraplegia during cross-clamping of the thoracic aorta
Effect of Spinal Cord Preconditioning on Paraplegia During Cross-Clamping of the Thoracic Aorta Katsuhiko Matsuyama, MD, Yukio Chiba, MD, Akio Ihaya, MD, Tetsuya Kimura, MD, Nobuhiko Tanigawa, MD, and Ryusuke Muraoka, MD Second Department of Surgery, Fukui Medical School, Yoshida-Gun, Fukui, Japan
Background. Paraplegia is a devastating complication of operations for thoracic or thoracoabdominal aneurysms. Preconditioning the brain with sublethal ischemia induces resistance to subsequent ordinarily lethal ischemia (ischemic tolerance), We investigated whether ischemic tolerance could be induced by preconditioning canine spinal cord. The role of heat-shock proteins (HSP) in this process was investigated. Methods. In experiment 1, the preconditioning group (n = 6) had aortic cross-clamping for 20 minutes, whereas controls (n = 6) had no cross-clamping. After 48 hours the aorta was cross-clamped for 60 minutes in both groups. Neurologic examination was performed 24 hours later and the spinal cord was studied for i m m u n o h i s t o c h e m i cally. In experiment 2, either 48 hours after 20 minutes of
clamping or after sham operation (n = 4), HSP were investigated immunohistochemically. Results. In experiment 1, 3 of 6 controls became paraplegic but none of the 6 preconditioning group dogs became paraplegic. The HSP appeared on sections from all 6 PC dogs and 3 control dogs that did not exhibit paraplegia. In experiment 2, HSP were present in clamped animals but could not be detected after sham operation. Conclusions. Ischemic tolerance was induced by preconditioning the canine spinal cord, in which HSP are believed to be involved.
paraplegia is a major complication of operations for thoracic or thoracoabdominal aneurysms [1], which appears related to spinal cord ischemia induced by low perfusion pressure during cross-clamping of the thoracic aorta. The reported incidence of paraplegia ranges from 3.8% to 17.6% [2]. Paraplegia is devastating and usually irreversible once it occurs. During the past decade, several attempts have been made experimentally and clinically to prevent paraplegia by the following methods: maintenance of distal aortic perfusion with shunts and bypasses [3], rapid identification and reimplantation of critical intercostal vessels [41, increasing spinal cord perfusion pressure with cerebrospinal fluid drainage [5], and administration of pharmacologic agents such as steroids [6], superoxide dismutase [7], and calcium blockers [8]. Somatosensory evoked potentials [9], evoked spinal cord potentials [10], and motor evoked potentials [11] have been monitored intraoperatively to detect spinal cord ischemia. Despite such efforts, no method has totally prevented the development of paraplegia. Kitagawa and colleagues [121 reported that preconditioning the brain with sublethal cerebral ischemia, which permits recovery without morphologically evident neuronal damage, induced resistance to subsequent ordi-
narily lethal period of ischemia in a gerbil model, a phenomenon called ischemic tolerance. Other experimental evidence indicates that prolonged but mild hypoperfusion and reversible oxidative stress have induced tolerance to subsequent lethal ischemia in gerbil hippocamal neurons [13, 14]. However, even a brief, nonlethal ischemic insult repeatedly administered at short intervals has been found to result in severe neuronal damage. To induce a neuroprotective effect, the interval between nonlethal ischemia and potentially lethal ischemia must be greater than I or 2 days, but not more than 14 [15]. Although a possible role of the stress response is suggested, the mechanism of acquisition of ischemic tolerance is currently unknown. One possible mechanism of the protective effects of preliminary exposures relates to the synthesis of heat-shock proteins (HSP) observed in gerbil [12] and rat models [161 of cerebral ischemia. Many experiments have been reported the effect of ischemic preconditioning in the brain. We investigated whether the ischemic tolerance phenomenon could be induced in the spinal cord by brief cross-clamping of the canine descending aorta. The HSP were also assessed immunohistochemically to determine whether they were induced by spinal ischemia.
F
Accepted for publication Nov 23, 1996. Address reprint requests to Dr Matsuyama, Second Department of Surgery, Fukui MedicalSchool,Matsuoka-Cho,Yoshida-Gun,Fukui-ken 910-11Japan. © 1997 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 1997;63:1315-20) © 1997 by The Society of Thoracic Surgeons
Material and Methods
All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH 0003-4975/97/517.00 PII S0003-4975(97)00104-5
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publication 85-23, revised 1985) and Guidelines for Animal Experiments, Fukui Medical School.
Experiment 1 Twelve adult beagle dogs weighing from 12 to 13 kg were anesthetized with intramuscular k e t a m i n e hydrochloride (10 m g / k g ) a n d i n t r a v e n o u s p e n t o b a r b i t a l s o d i u m (25 mg/kg) a n d p a r a l y z e d with p a n c u r o n i u m b r o m i d e (0.3 mg/kg). The trachea was i n t u b a t e d a n d ventilated on room air and low-flow oxygen (Harvard A p p a r a t u s Co, South Natick, MA). Respiratory settings included a tidal volume of 12 mL/kg, a respiratory rate to maintain the partial p r e s s u r e of carbon dioxide b e t w e e n 35 and 45 m m H g and that of oxygen above 100 m m Hg, as m e a s u r e d by arterial blood gas analysis (Ciba Corning Diagnostics Corp, Medfield, MA). A n a s o p h a r y n g e a l t e m p e r a t u r e p r o b e was inserted. The electrocardiogram was r e c o r d e d using needle electrodes. Catheters were inserted into the left carotid artery, a n d femoral artery to record the arterial pressure proximal a n d distal to aortic cross-clamping. All pressures were continuously monitored with a p r e s s u r e t r a n s d u c e r (P23XL, Nihon Koden, Tokyo, Japan). A left thoracotomy in the fourth intercostal space was p e r f o r m e d u n d e r sterile conditions. The aorta was isolated distal to the left subclavian artery and 100 U/kg of h e p a r i n was administered. The dogs were r a n d o m l y assigned to two experimental groups. The preconditioning operation (PC group: n - 6) included 20 m i n u t e s of aortic cross-clamping and the control operation (control group: n = 6) had no aortic cross-clamping. The aorta was cross-clamped distal to the left subclavian artery. No pharmacologic agents were used for control of blood pressure. After u n c l a m p i n g the aorta, 1 m g / k g of p r o t a m i n e was administered. The chest was closed in anatomic fashion a n d the animals were allowed to recover. All animals were noted to have a completely n o r m a l neurologic outcome. Reoperation was p e r f o r m e d 48 hours after the initial operation. U n d e r similar anesthesia, left thoracotomy was again p e r f o r m e d u n d e r sterile conditions. The aorta was c r o s s - c l a m p e d distal to the left subclavian artery for 60 minutes. Distal a n d proximal arterial p r e s s u r e were continuously monitored. No pharmacologic agents were used for control of blood pressure. The chest was closed in anatomic fashion, a n d the animals were allowed to recover. Neurologic examination of all animals was p e r f o r m e d by a investigator b l i n d e d to t r e a t m e n t group 24 hours after the second operation. The animals were g r a d e d according to a modification of the Tarlov classification [17], as follows: grade 0, spastic p a r a p l e g i a and no movem e n t of the lower limbs; grade 1, spastic p a r a p l e g i a a n d slight m o v e m e n t of the lower limbs; grade 2, good m o v e m e n t of the lower limbs but unable to stand; grade 3, able to stand but unable to walk normally; a n d grade 4, complete recovery. All animals were killed with an overdose of p e n t o b a r bital s o d i u m after neurologic assessment. The l u m b a r spinal cord was i m m e d i a t e l y r e m o v e d by posterior laminectomy after formalin perfusion fixation for i m m u n o histochemical analysis.
Ann Thorac Surg 1997;63:1315-20
Experiment 2 To investigate w h e t h e r HSP synthesis was i n d u c e d by ischemic preconditioning of the spinal cord, eight adult beagle dogs weighing b e t w e e n 12 a n d 13 kg were anesthetized, and four dogs received 20 minutes of aortic cross-clamping. For the control operation four dogs h a d no aortic cross-clamping. The animals were allow to recover for 48 hours after the surgical procedure. All animals then were killed with an overdose of p e n t o b a r bital sodium. The l u m b a r spinal cord was i m m e d i a t e l y r e m o v e d by posterior l a m i n e c t o m y after formalin perfusion fixation for i m m u n o h i s t o c h e m i c a l analysis a n d postfixed in 10% formalin for 1 week.
Immunohistochemical Studies After formalin fixation and e m b e d d i n g in paraffin, the spinal cord was serially sectioned using a microtome at a thickness of 5/zm. The sections were deparaffinized using xylene and g r a d e d alcohols. Endogenous peroxidase was inactivated using 3% h y d r o g e n peroxide in 0.05 mol/L p h o s p h a t e - b u f f e r e d saline for 10 minutes. Nonspecific protein b i n d i n g was blocked with normal bovine s e r u m for 10 minutes. This was followed b y an overnight incubation with a mouse monoclonal a n t i b o d y against HSP70 (SPA-810, Stressgen, Victoria, BC, Canada) diluted 1:200 at 4°C. The sections were then i n c u b a t e d with a biotinylated goat antimouse second antibody (LSAB kit; Dako Japan, Kyoto, Japan) and then visualized with d i a m i n o benzidine hydrochloride. The sections were counterstained with hematoxylin. Control sections were incub a t e d without the p r i m a r y antibody.
Statistical Analysis Each value was expressed as the m e a n + the s t a n d a r d deviation. Statistical evaluation was p e r f o r m e d by m e a n s of S t u d e n t ' s u n p a i r e d t test for comparison of e x p e r i m e n tal variables b e t w e e n the groups. The difference a m o n g groups in terms of the Tarlov scores was d e t e r m i n e d b y n o n p a r a m e t r i c statistical analysis u s i n g the M a n n W h i t n e y 13 test. A p value of less than 0.05 was considered significant.
Results
Experiment 1 HEMODYNAMIC MEASUREMENTS. There was no significant difference b e t w e e n the PC and the control groups with r e g a r d to e s o p h a g e a l temperatures, hemoglobin, h e m a t ocrit, pH, base excess, a n d preconditioning operation time (Table 1). Also, there was no statistically significant difference b e t w e e n the two groups with r e g a r d to proximal a n d distal m e a n arterial pressures during the baseline interval a n d reperfusion (Table 2). There was no significant difference b e t w e e n the two groups with regard to e s o p h a g e a l temperatures, hemoglobin, h e m a t o crit, pH, base excess, a n d operation time for 60 m i n u t e s of aortic cross-clamping (Table 3). Proximal m e a n arterial p r e s s u r e during cross-clamping was 172 +_ 9 m m Hg a n d 177 _+ 13 m m H g in PC a n d control groups, respectively. Distal mean arterial p r e s s u r e was 26 -+ 3 m m H g a n d 24 _+
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MATSUYAMAET AL SPINAL CORD PRECONDITIONING
Table 1. Variables in the Preconditioning Part of the Experiment" Characteristic Body weight (kg) Nasopharyngeal temperature (°C) Hemoglobin (g/dL) Hematocrit (%) Base excess pH Operation time (min)
Table 3. Variables at 60 Minutes of Aortic Cross-Clamping" Variable
PC
Control
12.6 _+ 0.6 36.6 + 0.8
12.4 -- 0.6 36.1 _+ 0.3
15.4 45.4 -1.9 7.388 70
+ 2.1 +_ 6.2 + 3.5 _+ 0.051 + 7
15.5 45.6 0.1 7.370 69
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-+ 2.3 = 6.6 = 3.7 ~ 0.064 z 14
Nasopharyngeal temperature (°C) Hemoglobin (g/dL) Hematocrit (%) Base excess pH Operation time (min) Tarlov score
PC
Control
36.8 + 0.3
36.8 _+ 0.6
15.1 +_ 1.4 43.5 +_ 6.3 2.8 ÷ 1.7 7.347 ÷ 0.055 112±9 3.8 _+ 0.4
16.0 _+ 2.1 46.4 ± 4.8 3.4 ÷ 1.5 7.370 + 0.030 115_+13 1.7 _+ 1.9b
Values represent mean -+ standard deviation, PC preconditioning.
Values represent mean _+ standard deviation. PC - preconditioning.
4 m m H g in PC a n d control groups, respectively. T h e r e was no significant difference b e t w e e n the two g r o u p s in distal a n d p r o x i m a l m e a n arterial p r e s s u r e d u r i n g 60 m i n u t e s of aortic c r o s s - c l a m p i n g (Table 4). NEUROLOGIC OUTCOME. A f t e r t h e initial o p e r a t i o n all anim a l s h a d a c o m p l e t e l y n o r m a l n e u r o l o g i c o u t c o m e . At 24 h o u r s after the s e c o n d o p e r a t i o n , 5 d o g s in the PC g r o u p h a d a c o m p l e t e l y n o r m a l n e u r o l o g i c o u t c o m e a n d 1 dog h a d a T a r l o v score of 3. T h r e e d o g s in the control g r o u p e x h i b i t e d paraplegia, 2 d o g s h a d a T a r l o v score of 3, a n d 1 dog had a completely normal neurologic outcome (Table 5). N e u r o l o g i c o u t c o m e was 3.8 _+ 0.4 a n d 1.7 ~ 1.9 in PC a n d control g r o u p s , respectively, r e p r e s e n t i n g a significant difference in Tarlov score b e t w e e n the two g r o u p s (Table 3). IMMUNOHISTOCHEMlSTRY. I m m u n o h i s t o c h e m i c a l s t a i n i n g u s i n g anti-HSP70 m o n o c l o n a l a n t i b o d y was p e r f o r m e d in the two groups. T h e H S P i m m u n o r e a c t i v i t y was o b s e r v e d on sections f r o m all 6 d o g s in the PC g r o u p a n d was also o b s e r v e d on sections f r o m 3 dogs that did not exhibit p a r a p l e g i a in the control group. In 9 n o n p a r a p l e g i c animals, i n c l u d i n g all 6 d o g s in the PC g r o u p a n d 3 dogs in t h e control group, n e u r o n s of the a n t e r i o r h o r n in the spinal cord w e r e s t a i n e d w i t h g r a n u l a r p a t t e r n in the cytoplasm. T h e glial cells also w e r e d e n s e l y s t a i n e d (Fig 1A). The sections f r o m 3 d o g s g r a d e d T a r l o v 0 in the control g r o u p r e v e a l e d s e v e r e d a m a g e i n c l u d i n g n e u r o nal d e g e n e r a t i o n , necrosis, a n d spongiosis. T h e H S P i m m u n o r e a c t i v i t y was not o b s e r v e d in the sections f r o m all 3 p a r a l y z e d d o g s (Fig 1B). N o specific i m m u n o r e a c t i v -
b p < 0.05.
ity was o b s e r v e d w h e n the p r i m a r y a n t i b o d y was o m i t ted.
Experiment 2 Histologic sections f r o m all 4 a n i m a l s killed 48 h o u r s after the control o p e r a t i o n did not s h o w any e v i d e n c e of H S P i m m u n o r e a c t i v i t y (Fig 1C). Sections f r o m a n i m a l s 48 h o u r s after the p r e c o n d i t i o n i n g o p e r a t i o n s h o w e d H S P i m m u n o r e a c t i v i t y in 3 of 4 dogs.
Comment Paraplegia, a m a j o r c o m p l i c a t i o n of o p e r a t i o n s for thoracic or t h o r a c o a b d o m i n a l a n e u r y s m s , is d e v a s t a t i n g a n d u s u a l l y irreversible. D e s p i t e v a r i o u s efforts, no m e t h o d has totally p r e v e n t e d t h e d e v e l o p m e n t of p a r a p l e g i a . K i t a g a w a a n d c o l l e a g u e s [12] h a v e r e p o r t e d that p r e c o n d i t i o n i n g the brain w i t h s u b l e t h a l c e r e b r a l i s c h e m i a i n d u c e d resistance to s u b s e q u e n t n o r m a l l y lethal p e r i o d of ischernia. This p h e n o m e n o n of i s c h e m i c t o l e r a n c e has b e e n d e s c r i b e d in b o t h rats a n d gerbils. In this s t u d y we i n v e s t i g a t e d w h e t h e r the i s c h e m i c t o l e r a n c e p h e n o m e n o n c o u l d be i n d u c e d by b r i e f crossc l a m p i n g of the c a n i n e d e s c e n d i n g aorta. W h e t h e r an i s c h e m i c insult is s u b l e t h a l or lethal d e p e n d s on distal or p r o x i m a l m e a n arterial p r e s s u r e a n d b o d y t e m p e r a t u r e d u r i n g aortic c r o s s - c l a m p i n g , as well as the d u r a t i o n of clamping. M a j o r r e p o r t s h a v e d e m o n s t r a t e d that p a r a p l e g i a in the d o g m o d e l occurs if the d u r a t i o n of aortic crossc l a m p i n g e x c e e d s 30 m i n u t e s [18, 19]. T h e r e f o r e , the aorta was c r o s s - c l a m p e d 20 m i n u t e s for i s c h e m i c p r e c o n d i t i o n i n g of spinal cord. In this study, distal a n d p r o x i m a l
Table 2. Mean Arterial Pressure in the Preconditioning Part of the Experiment (ram Hg)" Baseline Arterial Pressure Proximal Distal
During Cross-Clamping
Reperfusion
PC
Control
PC
Control
PC
Control
140 ± 12 137 _+ 15
144 _+ 6 137 - 15
172 ± 9 27 + 6
147 + 6 148 _+_6
139 ÷ 12 139 ~13
146 ± 10 138 _+ 15
Values represent mean z standard deviation. PC - preconditioning.
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Ann Thorac Surg 1997;63:1315-20
Table 4. Mean Arterial Pressure at 60 Minutes of Aortic Cross-Clamping (ram Hg)" Baseline Arterial Pressure Proximal Distal
Reperfusion
During Cross-Clamping
PC
Control
PC
Control
PC
Control
136 + 14 137 + 15
143 ± 6 144 _+ 7
172 ± 9 26 ± 3
177 _+ 13 24 + 4
139 + 17 138 ÷ 15
136 _+8 136 ± 8
Values represent mean -+ standard deviation. PC preconditioning.
m e a n arterial pressure were 165 +_ 12 m m Hg a n d 24 _+ 5 m m Hg, respectively. Twenty minutes of aortic crossc l a m p i n g was confirmed to be a sublethal insult to the spinal cord because all the animals h a d a completely normal neurologic outcome after the preconditioning operation. Kato a n d colleagues [15] r e p o r t e d that in the gerbil m o d e l of cerebral ischemia, 2 minutes of ischemia followed by 3 minutes of ischemia at intervals of 1, 2, 4, and 7 days caused a m a r k e d protective effect. Five-minute, 1-hour, a n d 6-hour intervals of the same ischemia resuited in almost complete neuronal damage, no protective effect was o b s e r v e d after 14 days. In our experiment, 60 minutes of spinal cord ischemia was given 48 hours after a 20-minute ischemia episode. Three of 6 animals in the control group b e c a m e paraplegic, but n o n e of 6 animals in the PC group b e c a m e paraplegic. There was a significant difference in Tarlov score b e t w e e n the PC and the control groups. This result d e m o n s t r a t e d that tolerance to lethal ischemia was i n d u c e d by preconditioning with a preceding p e r i o d of brief ischemia that did not itself p r o d u c e n e u r o n a l injury. Blaisdell a n d Cooley [20] r e p o r t e d that ligation of the first two pairs of intercostal arteries a n d occlusion of the d e s c e n d i n g aorta for 1 hour resulted in a 50% incidence of paraplegia. In our study, three of the six animals in the control group did not become paraplegic. This is p r e s u m ably attributable to individual differences in the anterior spinal circulatory pattern of the canine spinal cord even w h e n the aorta is cross-clamped for 60 minutes. No pharmacologic agents were used for the control of pressure to exclude their effects on the circulation. The m e c h a n i s m of acquisition of ischemic tolerance is currently unknown, although a role of the stress response is suggested. Recent studies have shown protein synthesis in the gerbil brain w h e n partially recovered 24 hours after a n d fully recovered 48 hours after preconditioning with 2 m i n u t e s of sublethal ischemia [21]. Various studies
Table 5. Neurologic Outcome at 24 Hours" Tarlov Score Group
0
1
2
3
4
PC Control
0 3
0 0
0 0
1 2
5 1
.1p < 0.05. PC = preconditioning.
have d e m o n s t r a t e d increased synthesis of stress proteins such as HSP70 after transient brain ischemia. A protective role against ischemic insult has b e e n h y p o t h e s i z e d for stress proteins [12]. Transient h y p e r t h e r m i a protects against s u b s e q u e n t forebrain ischemic cell d a m a g e in the rat [22] and the gerbil [23]. The HSP are a m o n g of the most frequently s t u d i e d stress proteins because of their relatively low level in n o n s t r e s s e d cells a n d dramatic elevation following stress [24]. The HSP synthesis increases w h e n cells are exposed to a variety of stresses including heat shock, viral infection, ischemia, trauma, and heavy metals, a n d are known to function as molecular c h a p e r o n e s in normal cellular processes [25] a n d are involved in facilitating protein folding, the a s s e m b l y of m a c r o m o l e c u l a r protein complexes, and protein traffic between intracellular c o m p a r t m e n t s [26]. The HSP act to ensure cell m e m b r a n e stability and facilitate d e n a t u r ation of irreversibly d a m a g e d proteins [27]. In these experiments, HSP a p p e a r e d in 3 n o n p a r a p l e gic dogs in the control group from e x p e r i m e n t 1. This suggests that the spinal cord ischemic insult due to 60 minutes of aortic cross-clamping was a sublethal stress of such a degree that HSP were induced. In e x p e r i m e n t 1, HSP a p p e a r e d in all nine n o n p a r a p l e g i c animals, but were not p r e s e n t in all 3 p a r a l y z e d animals. In experim e n t 2, HSP were not p r e s e n t in the s h a m - o p e r a t e d animals but a p p e a r e d in the p r e c o n d i t i o n i n g - o p e r a t e d animals. These results suggest that HSP are involved in the acquisition of ischemic tolerance in the spinal cord. There are several limitations to the present study. First, this is a canine model, a n d thus it m a y not necessarily be applicable to humans. The a n a t o m y of the blood s u p p l y to the canine spinal cord is also not the same as in humans. Second, this study had a very small s a m p l e size. Nevertheless, with this model, we have d e m o n s t r a t e d that spinal cord preconditioning significantly protects against paraplegia d u r i n g cross-clamping of the thoracic aorta. Third, the m e t h o d s used are impractical for clinical applications. The use of intraaortic balloon occlusion p e r c u t a n e o u s l y for spinal cord preconditioning has m a n y practical difficulties. The molecular m e c h a n i s m of the protective effect of HSP is not known, although it is r e p o r t e d that a b n o r m a l proteins serve as stress signals that trigger activation of HSP [28]. W h e t h e r HSP i n d u c e d after ischemic preconditioning directly play a protective role in n e u r o n s rem a i n s unknown. Further study is i m p o r t a n t because elucidation of the m e c h a n i s m m a y provide a clue for the prevention and t r e a t m e n t of p a r a p l e g i a in humans.
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References
A
Fig 1. lmmunohistochemical staining using anti-HSP70 antibody in the anterior horn of the lumbar spinal cord. (A) Histologic section from neurologically recovered animal in the preconditioning group. Immunoreactivity was seen with granular pattern in the cytoplasm of neurons. Also, the glial cells were densely stained. (B) Histologic section from paraplegic animal in the control group. Severe damage including neuronal degeneration, necrosis, and spongiosis was observed. No immunoreachvity was seen. (C) Histologic section from the animal 48 hours after the control operation. No staining was detected• (Original magnification qf all figures, ×200.)
1. Adams HD, Van Geertruyden HH. Neurological complication of aortic surgery. Ann Surg 1956;144:574-610. 2. Crawford ES, Rubio PA. Reappraisal of adjuncts to avoid ischemia in the treatment of aneurysms of descending thoracic aorta. J Thorac Cardiovasc Surg 1973;66:693-704. 3. Von Oppell UO, Dunne TT, De Groot KM, Zilla P. Spinal cord protection in the absence of collateral circulation: meta-analysis of mortality, and paraplegia. J Cardiol Surg 1994;9:685-91. 4. Svensson LG, Hess KR, Coselli JS, Sail HJ. Influence of segmental arteries, extent, and aortofemoral bypass on postoperative paraplegia after thoracoabdominal aortic operations. J Vasc Surg 1994;20:255-62. 5. Bower GT, Murray MJ, Gloviczki P, et al. Effects of thoracic occlusion and cerebrospinal fluid drainage on regional spinal cord blood flow in dogs: correlation with neurologic outcome. J Vasc Surg 1989;9:135-44. 6. Laschinger JC, Cunningham JN Jr, Cooper MM, Krieger K, Nathan IM, Spencer FC. Prevention of ischemic spinal cord injury following aortic cross-clamping. Use of corticosteroids. Ann Thorac Surg 1984;38:500-7. 7. Granke K, Hollier LH, Zdrahal P, Moore W. Longitudinal study of cerebral spinal fluid drainage in polyethylene glycol-conjugated superoxide dismutase in paraplegia associated with thoracic aortic cross-clamping. J Vasc Surg 1991; 13:615-21. 8. De Mol B, Hamerlijnck R, Janssen T, Jageneau A. Infrarenal aortic occlusion in the rabbit to assess the effect of flunarizine in the prevention of ischemic spinal cord injury. Thorac Cardiovasc Surg 1991;39:36-9. 9. Grabitz K, Sandmann W, Stuhmeier K, et al. The risk of ischemic spinal cord injury in patients undergoing graft replacement for thoracoabdominal aortic aneurysms. J Vasc Surg 1996;23:230-40. 10. Yamamoto N, Takano H, Kitagawa H, Kawaguchi Y, Tsuji H, Uozaki Y. Monitoring for spinal cord ischemia by use of the evoked spinal cord potentials during aortic aneurysm surgery. J Vasc Surg 1994;20:826-33. 11. Reuter DG, Tacker WA Jr, Badylak SF, Voorhees WD III, Konrad PE. Correlation of motor-evoked potential response to ischemic spinal cord damage. J Thorac Cardiovasc Surg 1992;104:262-72. 12. Kitagawa K, Matsumoto M, Tagaya M, et al. 'Ischemic tolerance' phenomenon found in the brain. Brain Res 1990; 528:21-4. 13. Ohtuki T, Matsumoto M, Kitagawa K, et al. Induced resistance and susceptibility to cerebral ischemia in gerbil hippocamal neurons by prolonged but mild hypoperfusion. Brain Res 1993;614:279-84• 14. Ohtuki T, Matsumoto M, Kuwabara K, et al. Influence of oxidative stress on induced tolerance to ischemia in gerbil hippocamal neurons. Brain Res 1992;599:246-52. 15. Kato H, Liu Y, Araki T, Kogure K. Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects• Brain Res 1991; 553:238- 42. 16. Nishi S, Taki W, Uemura Y, et al. Ischemic tolerance due to the induction of HSP70 in a rat ischemic recirculation model. Brain Res 1993;615:281-8. 17. Tarlov IM. Spinal cord compression: mechanisms of paralysis and treatment. Springfield, IL: Charles C. Thomas, 1957;147. 18. Mutch WA, Graham MR, Halliday WC, Thiessen DB, Girling LG. Use of neuroanesthesia adjuncts (hyperventilation and mannitol administration) improves neurological outcome after thoracic aortic cross-clamping in dogs. Stroke 1993;24: 1204-11. 19. Woloszyn TT, Marini CP, Coons MS, et al. Cerebrospinal fluid drainage and steroids provide better spinal cord protection during aortic cross-clamping than does either treatment alone. Ann Thorac Surg 1990;49:78-83.
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20. Blaisdell FW, Cooley DA. The mechanism of paraplegia after temporary thoracic aortic occlusion and its relationship to spinal fluid pressure. Ann Thorac Surg 1962;51: 351-5. 21. Kato H, Kogure K, Nakata N, Araki T, Itoyama Y. Facilitated recovery from postischemic suppression of protein synthesis in the gerbil with ischemic tolerance. Brain Research Bull 1995;36:205- 8. 22. Chopp M, Chen H, Ho KL, et al. Transient hyperthermia protects against subsequent forebrain ischemic cell damage in the rat. Neurology 1989;39:1396-8. 23. Kitagawa K, Matsumoto M, Tagaya M, et al. Hyperthermiainduced neuronal protection against ischemic injury in gerbils. J Cereb Blood Flow Metab 1991;11:449-52.
Ann Thorac Surg 1997;63:1315-20
24. Voellmy R, Ahmed A, Schiller P, Bromley P, Rungger D. Isolation and functional analysis of a human 70,000-dalton heat shock protein gene segment. Proc Natl Acad USA 1985; 82:4949-53. 25. Lindquist S. The heat shock response. Annu Rev Biochem 1986;55:1151-91. 26. Gething MJ, Sambrook J. Protein folding in the cell. Nature 1992;355:33-45. 27. Chiang HL, Terlecky SR, Plant CP, Dice JF. A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Science 1989;246:382-5. 28. Ananthan J , Goldberg AlL, Voelimy R. Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 1986;232:522-4.
Important News: New HCFA Crisis Update Section on the STS Web Site Recently the H e a l t h Care F i n a n c i n g A d m i n i s t r a t i o n (HCFA) has p r o p o s e d crippling reductions in Medicare Fee Schedules for cardiothoracic s u r g e r y - - r e d u c t i o n s that threaten our very ability to deliver health care to the vast Medicare population. The Council and m e m b e r s h i p of The Society of Thoracic Surgeons (STS) are r e s p o n d i n g in crisis mode, mobilizing efforts in every possible direction to o p p o s e this decision. As STS President George C. Kaiser e m p h a s i z e d in his recent m e m o r a n d u m , "Call to Action," "The seriousness of this p r o b l e m dictates that all cardiothoracic surgeons r e s p o n d i m m e d i a t e l y a n d decisively to this threat to disrupt the finest healthcare system ever created." A n e w " H C F A Crisis U p d a t e " section has b e e n created on the STS W e b site as a communications center to u p d a t e our m e m b e r s h i p , and any other interested parties, on these issues. This site contains important documents including details of the HCFA proposal, the STS position paper, the "Call to Action," the STS Resolution to form a PAC, and key letters to HCFA. A n on-line
© 1997 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
discussion has been started, a n d any helpful ideas or suggestions are needed. STS M e m b e r s are u r g e d to write their m e m b e r s of Congress; an STS m e m o is being p r e p a r e d suggesting key points. Robert Wilbur, STS representative in W a s h ington, DC, e m p h a s i z e s that "Letters on p a p e r are much better, and they should be individualized, not boilerplate. Follow-up on E-mail is fine after the letter, b u t letter first, please. STS m e m b e r s who know their m e m b e r of Congress should telephone. W e are a l r e a d y having positive response from t e l e p h o n e calls. Describe the basics of the p r o b l e m and ask the C o n g r e s s p e r s o n ' s office to contact me for m o r e detailed information. I have had several very good meetings with Congressional staff this w e e k following on contact from STS m e m b e r s . " The STS W e b site can help y o u - - s e e "Find Your M e m b e r of C o n g r e s s " on the HCFA Crisis U p d a t e page, at http://www.sts.org. Here is a practical chance for each of us to influence the i m m e d i a t e future of our profession.
Ann Thorac Surg 1997;63:1320 • 0003-4975/97/$17.00 PII S0003-4975(97)00406-2
Background. Paraplegia is a devastating complication of operations for thoracic or thoracoabdominal aneurysms. Preconditioning the brain with sublethal ischemia induces resistance to subsequent ordinarily lethal ischemia (ischemic tolerance), We investigated whether ischemic tolerance could be induced by preconditioning canine spinal cord. The role of heat-shock proteins (HSP) in this process was investigated. Methods. In experiment 1, the preconditioning group (n = 6) had aortic cross-clamping for 20 minutes, whereas controls (n = 6) had no cross-clamping. After 48 hours the aorta was cross-clamped for 60 minutes in both groups. Neurologic examination was performed 24 hours later and the spinal cord was studied for i m m u n o h i s t o c h e m i cally. In experiment 2, either 48 hours after 20 minutes of
clamping or after sham operation (n = 4), HSP were investigated immunohistochemically. Results. In experiment 1, 3 of 6 controls became paraplegic but none of the 6 preconditioning group dogs became paraplegic. The HSP appeared on sections from all 6 PC dogs and 3 control dogs that did not exhibit paraplegia. In experiment 2, HSP were present in clamped animals but could not be detected after sham operation. Conclusions. Ischemic tolerance was induced by preconditioning the canine spinal cord, in which HSP are believed to be involved.
paraplegia is a major complication of operations for thoracic or thoracoabdominal aneurysms [1], which appears related to spinal cord ischemia induced by low perfusion pressure during cross-clamping of the thoracic aorta. The reported incidence of paraplegia ranges from 3.8% to 17.6% [2]. Paraplegia is devastating and usually irreversible once it occurs. During the past decade, several attempts have been made experimentally and clinically to prevent paraplegia by the following methods: maintenance of distal aortic perfusion with shunts and bypasses [3], rapid identification and reimplantation of critical intercostal vessels [41, increasing spinal cord perfusion pressure with cerebrospinal fluid drainage [5], and administration of pharmacologic agents such as steroids [6], superoxide dismutase [7], and calcium blockers [8]. Somatosensory evoked potentials [9], evoked spinal cord potentials [10], and motor evoked potentials [11] have been monitored intraoperatively to detect spinal cord ischemia. Despite such efforts, no method has totally prevented the development of paraplegia. Kitagawa and colleagues [121 reported that preconditioning the brain with sublethal cerebral ischemia, which permits recovery without morphologically evident neuronal damage, induced resistance to subsequent ordi-
narily lethal period of ischemia in a gerbil model, a phenomenon called ischemic tolerance. Other experimental evidence indicates that prolonged but mild hypoperfusion and reversible oxidative stress have induced tolerance to subsequent lethal ischemia in gerbil hippocamal neurons [13, 14]. However, even a brief, nonlethal ischemic insult repeatedly administered at short intervals has been found to result in severe neuronal damage. To induce a neuroprotective effect, the interval between nonlethal ischemia and potentially lethal ischemia must be greater than I or 2 days, but not more than 14 [15]. Although a possible role of the stress response is suggested, the mechanism of acquisition of ischemic tolerance is currently unknown. One possible mechanism of the protective effects of preliminary exposures relates to the synthesis of heat-shock proteins (HSP) observed in gerbil [12] and rat models [161 of cerebral ischemia. Many experiments have been reported the effect of ischemic preconditioning in the brain. We investigated whether the ischemic tolerance phenomenon could be induced in the spinal cord by brief cross-clamping of the canine descending aorta. The HSP were also assessed immunohistochemically to determine whether they were induced by spinal ischemia.
F
Accepted for publication Nov 23, 1996. Address reprint requests to Dr Matsuyama, Second Department of Surgery, Fukui MedicalSchool,Matsuoka-Cho,Yoshida-Gun,Fukui-ken 910-11Japan. © 1997 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 1997;63:1315-20) © 1997 by The Society of Thoracic Surgeons
Material and Methods
All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH 0003-4975/97/517.00 PII S0003-4975(97)00104-5
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MATSUYAMA ET AL SPINAL CORD PRECONDITIONING
publication 85-23, revised 1985) and Guidelines for Animal Experiments, Fukui Medical School.
Experiment 1 Twelve adult beagle dogs weighing from 12 to 13 kg were anesthetized with intramuscular k e t a m i n e hydrochloride (10 m g / k g ) a n d i n t r a v e n o u s p e n t o b a r b i t a l s o d i u m (25 mg/kg) a n d p a r a l y z e d with p a n c u r o n i u m b r o m i d e (0.3 mg/kg). The trachea was i n t u b a t e d a n d ventilated on room air and low-flow oxygen (Harvard A p p a r a t u s Co, South Natick, MA). Respiratory settings included a tidal volume of 12 mL/kg, a respiratory rate to maintain the partial p r e s s u r e of carbon dioxide b e t w e e n 35 and 45 m m H g and that of oxygen above 100 m m Hg, as m e a s u r e d by arterial blood gas analysis (Ciba Corning Diagnostics Corp, Medfield, MA). A n a s o p h a r y n g e a l t e m p e r a t u r e p r o b e was inserted. The electrocardiogram was r e c o r d e d using needle electrodes. Catheters were inserted into the left carotid artery, a n d femoral artery to record the arterial pressure proximal a n d distal to aortic cross-clamping. All pressures were continuously monitored with a p r e s s u r e t r a n s d u c e r (P23XL, Nihon Koden, Tokyo, Japan). A left thoracotomy in the fourth intercostal space was p e r f o r m e d u n d e r sterile conditions. The aorta was isolated distal to the left subclavian artery and 100 U/kg of h e p a r i n was administered. The dogs were r a n d o m l y assigned to two experimental groups. The preconditioning operation (PC group: n - 6) included 20 m i n u t e s of aortic cross-clamping and the control operation (control group: n = 6) had no aortic cross-clamping. The aorta was cross-clamped distal to the left subclavian artery. No pharmacologic agents were used for control of blood pressure. After u n c l a m p i n g the aorta, 1 m g / k g of p r o t a m i n e was administered. The chest was closed in anatomic fashion a n d the animals were allowed to recover. All animals were noted to have a completely n o r m a l neurologic outcome. Reoperation was p e r f o r m e d 48 hours after the initial operation. U n d e r similar anesthesia, left thoracotomy was again p e r f o r m e d u n d e r sterile conditions. The aorta was c r o s s - c l a m p e d distal to the left subclavian artery for 60 minutes. Distal a n d proximal arterial p r e s s u r e were continuously monitored. No pharmacologic agents were used for control of blood pressure. The chest was closed in anatomic fashion, a n d the animals were allowed to recover. Neurologic examination of all animals was p e r f o r m e d by a investigator b l i n d e d to t r e a t m e n t group 24 hours after the second operation. The animals were g r a d e d according to a modification of the Tarlov classification [17], as follows: grade 0, spastic p a r a p l e g i a and no movem e n t of the lower limbs; grade 1, spastic p a r a p l e g i a a n d slight m o v e m e n t of the lower limbs; grade 2, good m o v e m e n t of the lower limbs but unable to stand; grade 3, able to stand but unable to walk normally; a n d grade 4, complete recovery. All animals were killed with an overdose of p e n t o b a r bital s o d i u m after neurologic assessment. The l u m b a r spinal cord was i m m e d i a t e l y r e m o v e d by posterior laminectomy after formalin perfusion fixation for i m m u n o histochemical analysis.
Ann Thorac Surg 1997;63:1315-20
Experiment 2 To investigate w h e t h e r HSP synthesis was i n d u c e d by ischemic preconditioning of the spinal cord, eight adult beagle dogs weighing b e t w e e n 12 a n d 13 kg were anesthetized, and four dogs received 20 minutes of aortic cross-clamping. For the control operation four dogs h a d no aortic cross-clamping. The animals were allow to recover for 48 hours after the surgical procedure. All animals then were killed with an overdose of p e n t o b a r bital sodium. The l u m b a r spinal cord was i m m e d i a t e l y r e m o v e d by posterior l a m i n e c t o m y after formalin perfusion fixation for i m m u n o h i s t o c h e m i c a l analysis a n d postfixed in 10% formalin for 1 week.
Immunohistochemical Studies After formalin fixation and e m b e d d i n g in paraffin, the spinal cord was serially sectioned using a microtome at a thickness of 5/zm. The sections were deparaffinized using xylene and g r a d e d alcohols. Endogenous peroxidase was inactivated using 3% h y d r o g e n peroxide in 0.05 mol/L p h o s p h a t e - b u f f e r e d saline for 10 minutes. Nonspecific protein b i n d i n g was blocked with normal bovine s e r u m for 10 minutes. This was followed b y an overnight incubation with a mouse monoclonal a n t i b o d y against HSP70 (SPA-810, Stressgen, Victoria, BC, Canada) diluted 1:200 at 4°C. The sections were then i n c u b a t e d with a biotinylated goat antimouse second antibody (LSAB kit; Dako Japan, Kyoto, Japan) and then visualized with d i a m i n o benzidine hydrochloride. The sections were counterstained with hematoxylin. Control sections were incub a t e d without the p r i m a r y antibody.
Statistical Analysis Each value was expressed as the m e a n + the s t a n d a r d deviation. Statistical evaluation was p e r f o r m e d by m e a n s of S t u d e n t ' s u n p a i r e d t test for comparison of e x p e r i m e n tal variables b e t w e e n the groups. The difference a m o n g groups in terms of the Tarlov scores was d e t e r m i n e d b y n o n p a r a m e t r i c statistical analysis u s i n g the M a n n W h i t n e y 13 test. A p value of less than 0.05 was considered significant.
Results
Experiment 1 HEMODYNAMIC MEASUREMENTS. There was no significant difference b e t w e e n the PC and the control groups with r e g a r d to e s o p h a g e a l temperatures, hemoglobin, h e m a t ocrit, pH, base excess, a n d preconditioning operation time (Table 1). Also, there was no statistically significant difference b e t w e e n the two groups with r e g a r d to proximal a n d distal m e a n arterial pressures during the baseline interval a n d reperfusion (Table 2). There was no significant difference b e t w e e n the two groups with regard to e s o p h a g e a l temperatures, hemoglobin, h e m a t o crit, pH, base excess, a n d operation time for 60 m i n u t e s of aortic cross-clamping (Table 3). Proximal m e a n arterial p r e s s u r e during cross-clamping was 172 +_ 9 m m Hg a n d 177 _+ 13 m m H g in PC a n d control groups, respectively. Distal mean arterial p r e s s u r e was 26 -+ 3 m m H g a n d 24 _+
Ann Thorac Surg 1997;63:1315-20
MATSUYAMAET AL SPINAL CORD PRECONDITIONING
Table 1. Variables in the Preconditioning Part of the Experiment" Characteristic Body weight (kg) Nasopharyngeal temperature (°C) Hemoglobin (g/dL) Hematocrit (%) Base excess pH Operation time (min)
Table 3. Variables at 60 Minutes of Aortic Cross-Clamping" Variable
PC
Control
12.6 _+ 0.6 36.6 + 0.8
12.4 -- 0.6 36.1 _+ 0.3
15.4 45.4 -1.9 7.388 70
+ 2.1 +_ 6.2 + 3.5 _+ 0.051 + 7
15.5 45.6 0.1 7.370 69
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-+ 2.3 = 6.6 = 3.7 ~ 0.064 z 14
Nasopharyngeal temperature (°C) Hemoglobin (g/dL) Hematocrit (%) Base excess pH Operation time (min) Tarlov score
PC
Control
36.8 + 0.3
36.8 _+ 0.6
15.1 +_ 1.4 43.5 +_ 6.3 2.8 ÷ 1.7 7.347 ÷ 0.055 112±9 3.8 _+ 0.4
16.0 _+ 2.1 46.4 ± 4.8 3.4 ÷ 1.5 7.370 + 0.030 115_+13 1.7 _+ 1.9b
Values represent mean -+ standard deviation, PC preconditioning.
Values represent mean _+ standard deviation. PC - preconditioning.
4 m m H g in PC a n d control groups, respectively. T h e r e was no significant difference b e t w e e n the two g r o u p s in distal a n d p r o x i m a l m e a n arterial p r e s s u r e d u r i n g 60 m i n u t e s of aortic c r o s s - c l a m p i n g (Table 4). NEUROLOGIC OUTCOME. A f t e r t h e initial o p e r a t i o n all anim a l s h a d a c o m p l e t e l y n o r m a l n e u r o l o g i c o u t c o m e . At 24 h o u r s after the s e c o n d o p e r a t i o n , 5 d o g s in the PC g r o u p h a d a c o m p l e t e l y n o r m a l n e u r o l o g i c o u t c o m e a n d 1 dog h a d a T a r l o v score of 3. T h r e e d o g s in the control g r o u p e x h i b i t e d paraplegia, 2 d o g s h a d a T a r l o v score of 3, a n d 1 dog had a completely normal neurologic outcome (Table 5). N e u r o l o g i c o u t c o m e was 3.8 _+ 0.4 a n d 1.7 ~ 1.9 in PC a n d control g r o u p s , respectively, r e p r e s e n t i n g a significant difference in Tarlov score b e t w e e n the two g r o u p s (Table 3). IMMUNOHISTOCHEMlSTRY. I m m u n o h i s t o c h e m i c a l s t a i n i n g u s i n g anti-HSP70 m o n o c l o n a l a n t i b o d y was p e r f o r m e d in the two groups. T h e H S P i m m u n o r e a c t i v i t y was o b s e r v e d on sections f r o m all 6 d o g s in the PC g r o u p a n d was also o b s e r v e d on sections f r o m 3 dogs that did not exhibit p a r a p l e g i a in the control group. In 9 n o n p a r a p l e g i c animals, i n c l u d i n g all 6 d o g s in the PC g r o u p a n d 3 dogs in t h e control group, n e u r o n s of the a n t e r i o r h o r n in the spinal cord w e r e s t a i n e d w i t h g r a n u l a r p a t t e r n in the cytoplasm. T h e glial cells also w e r e d e n s e l y s t a i n e d (Fig 1A). The sections f r o m 3 d o g s g r a d e d T a r l o v 0 in the control g r o u p r e v e a l e d s e v e r e d a m a g e i n c l u d i n g n e u r o nal d e g e n e r a t i o n , necrosis, a n d spongiosis. T h e H S P i m m u n o r e a c t i v i t y was not o b s e r v e d in the sections f r o m all 3 p a r a l y z e d d o g s (Fig 1B). N o specific i m m u n o r e a c t i v -
b p < 0.05.
ity was o b s e r v e d w h e n the p r i m a r y a n t i b o d y was o m i t ted.
Experiment 2 Histologic sections f r o m all 4 a n i m a l s killed 48 h o u r s after the control o p e r a t i o n did not s h o w any e v i d e n c e of H S P i m m u n o r e a c t i v i t y (Fig 1C). Sections f r o m a n i m a l s 48 h o u r s after the p r e c o n d i t i o n i n g o p e r a t i o n s h o w e d H S P i m m u n o r e a c t i v i t y in 3 of 4 dogs.
Comment Paraplegia, a m a j o r c o m p l i c a t i o n of o p e r a t i o n s for thoracic or t h o r a c o a b d o m i n a l a n e u r y s m s , is d e v a s t a t i n g a n d u s u a l l y irreversible. D e s p i t e v a r i o u s efforts, no m e t h o d has totally p r e v e n t e d t h e d e v e l o p m e n t of p a r a p l e g i a . K i t a g a w a a n d c o l l e a g u e s [12] h a v e r e p o r t e d that p r e c o n d i t i o n i n g the brain w i t h s u b l e t h a l c e r e b r a l i s c h e m i a i n d u c e d resistance to s u b s e q u e n t n o r m a l l y lethal p e r i o d of ischernia. This p h e n o m e n o n of i s c h e m i c t o l e r a n c e has b e e n d e s c r i b e d in b o t h rats a n d gerbils. In this s t u d y we i n v e s t i g a t e d w h e t h e r the i s c h e m i c t o l e r a n c e p h e n o m e n o n c o u l d be i n d u c e d by b r i e f crossc l a m p i n g of the c a n i n e d e s c e n d i n g aorta. W h e t h e r an i s c h e m i c insult is s u b l e t h a l or lethal d e p e n d s on distal or p r o x i m a l m e a n arterial p r e s s u r e a n d b o d y t e m p e r a t u r e d u r i n g aortic c r o s s - c l a m p i n g , as well as the d u r a t i o n of clamping. M a j o r r e p o r t s h a v e d e m o n s t r a t e d that p a r a p l e g i a in the d o g m o d e l occurs if the d u r a t i o n of aortic crossc l a m p i n g e x c e e d s 30 m i n u t e s [18, 19]. T h e r e f o r e , the aorta was c r o s s - c l a m p e d 20 m i n u t e s for i s c h e m i c p r e c o n d i t i o n i n g of spinal cord. In this study, distal a n d p r o x i m a l
Table 2. Mean Arterial Pressure in the Preconditioning Part of the Experiment (ram Hg)" Baseline Arterial Pressure Proximal Distal
During Cross-Clamping
Reperfusion
PC
Control
PC
Control
PC
Control
140 ± 12 137 _+ 15
144 _+ 6 137 - 15
172 ± 9 27 + 6
147 + 6 148 _+_6
139 ÷ 12 139 ~13
146 ± 10 138 _+ 15
Values represent mean z standard deviation. PC - preconditioning.
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MATSUYAMAET AL SPINAL CORD PRECONDITIONING
Ann Thorac Surg 1997;63:1315-20
Table 4. Mean Arterial Pressure at 60 Minutes of Aortic Cross-Clamping (ram Hg)" Baseline Arterial Pressure Proximal Distal
Reperfusion
During Cross-Clamping
PC
Control
PC
Control
PC
Control
136 + 14 137 + 15
143 ± 6 144 _+ 7
172 ± 9 26 ± 3
177 _+ 13 24 + 4
139 + 17 138 ÷ 15
136 _+8 136 ± 8
Values represent mean -+ standard deviation. PC preconditioning.
m e a n arterial pressure were 165 +_ 12 m m Hg a n d 24 _+ 5 m m Hg, respectively. Twenty minutes of aortic crossc l a m p i n g was confirmed to be a sublethal insult to the spinal cord because all the animals h a d a completely normal neurologic outcome after the preconditioning operation. Kato a n d colleagues [15] r e p o r t e d that in the gerbil m o d e l of cerebral ischemia, 2 minutes of ischemia followed by 3 minutes of ischemia at intervals of 1, 2, 4, and 7 days caused a m a r k e d protective effect. Five-minute, 1-hour, a n d 6-hour intervals of the same ischemia resuited in almost complete neuronal damage, no protective effect was o b s e r v e d after 14 days. In our experiment, 60 minutes of spinal cord ischemia was given 48 hours after a 20-minute ischemia episode. Three of 6 animals in the control group b e c a m e paraplegic, but n o n e of 6 animals in the PC group b e c a m e paraplegic. There was a significant difference in Tarlov score b e t w e e n the PC and the control groups. This result d e m o n s t r a t e d that tolerance to lethal ischemia was i n d u c e d by preconditioning with a preceding p e r i o d of brief ischemia that did not itself p r o d u c e n e u r o n a l injury. Blaisdell a n d Cooley [20] r e p o r t e d that ligation of the first two pairs of intercostal arteries a n d occlusion of the d e s c e n d i n g aorta for 1 hour resulted in a 50% incidence of paraplegia. In our study, three of the six animals in the control group did not become paraplegic. This is p r e s u m ably attributable to individual differences in the anterior spinal circulatory pattern of the canine spinal cord even w h e n the aorta is cross-clamped for 60 minutes. No pharmacologic agents were used for the control of pressure to exclude their effects on the circulation. The m e c h a n i s m of acquisition of ischemic tolerance is currently unknown, although a role of the stress response is suggested. Recent studies have shown protein synthesis in the gerbil brain w h e n partially recovered 24 hours after a n d fully recovered 48 hours after preconditioning with 2 m i n u t e s of sublethal ischemia [21]. Various studies
Table 5. Neurologic Outcome at 24 Hours" Tarlov Score Group
0
1
2
3
4
PC Control
0 3
0 0
0 0
1 2
5 1
.1p < 0.05. PC = preconditioning.
have d e m o n s t r a t e d increased synthesis of stress proteins such as HSP70 after transient brain ischemia. A protective role against ischemic insult has b e e n h y p o t h e s i z e d for stress proteins [12]. Transient h y p e r t h e r m i a protects against s u b s e q u e n t forebrain ischemic cell d a m a g e in the rat [22] and the gerbil [23]. The HSP are a m o n g of the most frequently s t u d i e d stress proteins because of their relatively low level in n o n s t r e s s e d cells a n d dramatic elevation following stress [24]. The HSP synthesis increases w h e n cells are exposed to a variety of stresses including heat shock, viral infection, ischemia, trauma, and heavy metals, a n d are known to function as molecular c h a p e r o n e s in normal cellular processes [25] a n d are involved in facilitating protein folding, the a s s e m b l y of m a c r o m o l e c u l a r protein complexes, and protein traffic between intracellular c o m p a r t m e n t s [26]. The HSP act to ensure cell m e m b r a n e stability and facilitate d e n a t u r ation of irreversibly d a m a g e d proteins [27]. In these experiments, HSP a p p e a r e d in 3 n o n p a r a p l e gic dogs in the control group from e x p e r i m e n t 1. This suggests that the spinal cord ischemic insult due to 60 minutes of aortic cross-clamping was a sublethal stress of such a degree that HSP were induced. In e x p e r i m e n t 1, HSP a p p e a r e d in all nine n o n p a r a p l e g i c animals, but were not p r e s e n t in all 3 p a r a l y z e d animals. In experim e n t 2, HSP were not p r e s e n t in the s h a m - o p e r a t e d animals but a p p e a r e d in the p r e c o n d i t i o n i n g - o p e r a t e d animals. These results suggest that HSP are involved in the acquisition of ischemic tolerance in the spinal cord. There are several limitations to the present study. First, this is a canine model, a n d thus it m a y not necessarily be applicable to humans. The a n a t o m y of the blood s u p p l y to the canine spinal cord is also not the same as in humans. Second, this study had a very small s a m p l e size. Nevertheless, with this model, we have d e m o n s t r a t e d that spinal cord preconditioning significantly protects against paraplegia d u r i n g cross-clamping of the thoracic aorta. Third, the m e t h o d s used are impractical for clinical applications. The use of intraaortic balloon occlusion p e r c u t a n e o u s l y for spinal cord preconditioning has m a n y practical difficulties. The molecular m e c h a n i s m of the protective effect of HSP is not known, although it is r e p o r t e d that a b n o r m a l proteins serve as stress signals that trigger activation of HSP [28]. W h e t h e r HSP i n d u c e d after ischemic preconditioning directly play a protective role in n e u r o n s rem a i n s unknown. Further study is i m p o r t a n t because elucidation of the m e c h a n i s m m a y provide a clue for the prevention and t r e a t m e n t of p a r a p l e g i a in humans.
Ann Thorac Surg 1997;63:1315-20
MATSUYAMAET AL SPINAL CORD PRECONDITIONING
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References
A
Fig 1. lmmunohistochemical staining using anti-HSP70 antibody in the anterior horn of the lumbar spinal cord. (A) Histologic section from neurologically recovered animal in the preconditioning group. Immunoreactivity was seen with granular pattern in the cytoplasm of neurons. Also, the glial cells were densely stained. (B) Histologic section from paraplegic animal in the control group. Severe damage including neuronal degeneration, necrosis, and spongiosis was observed. No immunoreachvity was seen. (C) Histologic section from the animal 48 hours after the control operation. No staining was detected• (Original magnification qf all figures, ×200.)
1. Adams HD, Van Geertruyden HH. Neurological complication of aortic surgery. Ann Surg 1956;144:574-610. 2. Crawford ES, Rubio PA. Reappraisal of adjuncts to avoid ischemia in the treatment of aneurysms of descending thoracic aorta. J Thorac Cardiovasc Surg 1973;66:693-704. 3. Von Oppell UO, Dunne TT, De Groot KM, Zilla P. Spinal cord protection in the absence of collateral circulation: meta-analysis of mortality, and paraplegia. J Cardiol Surg 1994;9:685-91. 4. Svensson LG, Hess KR, Coselli JS, Sail HJ. Influence of segmental arteries, extent, and aortofemoral bypass on postoperative paraplegia after thoracoabdominal aortic operations. J Vasc Surg 1994;20:255-62. 5. Bower GT, Murray MJ, Gloviczki P, et al. Effects of thoracic occlusion and cerebrospinal fluid drainage on regional spinal cord blood flow in dogs: correlation with neurologic outcome. J Vasc Surg 1989;9:135-44. 6. Laschinger JC, Cunningham JN Jr, Cooper MM, Krieger K, Nathan IM, Spencer FC. Prevention of ischemic spinal cord injury following aortic cross-clamping. Use of corticosteroids. Ann Thorac Surg 1984;38:500-7. 7. Granke K, Hollier LH, Zdrahal P, Moore W. Longitudinal study of cerebral spinal fluid drainage in polyethylene glycol-conjugated superoxide dismutase in paraplegia associated with thoracic aortic cross-clamping. J Vasc Surg 1991; 13:615-21. 8. De Mol B, Hamerlijnck R, Janssen T, Jageneau A. Infrarenal aortic occlusion in the rabbit to assess the effect of flunarizine in the prevention of ischemic spinal cord injury. Thorac Cardiovasc Surg 1991;39:36-9. 9. Grabitz K, Sandmann W, Stuhmeier K, et al. The risk of ischemic spinal cord injury in patients undergoing graft replacement for thoracoabdominal aortic aneurysms. J Vasc Surg 1996;23:230-40. 10. Yamamoto N, Takano H, Kitagawa H, Kawaguchi Y, Tsuji H, Uozaki Y. Monitoring for spinal cord ischemia by use of the evoked spinal cord potentials during aortic aneurysm surgery. J Vasc Surg 1994;20:826-33. 11. Reuter DG, Tacker WA Jr, Badylak SF, Voorhees WD III, Konrad PE. Correlation of motor-evoked potential response to ischemic spinal cord damage. J Thorac Cardiovasc Surg 1992;104:262-72. 12. Kitagawa K, Matsumoto M, Tagaya M, et al. 'Ischemic tolerance' phenomenon found in the brain. Brain Res 1990; 528:21-4. 13. Ohtuki T, Matsumoto M, Kitagawa K, et al. Induced resistance and susceptibility to cerebral ischemia in gerbil hippocamal neurons by prolonged but mild hypoperfusion. Brain Res 1993;614:279-84• 14. Ohtuki T, Matsumoto M, Kuwabara K, et al. Influence of oxidative stress on induced tolerance to ischemia in gerbil hippocamal neurons. Brain Res 1992;599:246-52. 15. Kato H, Liu Y, Araki T, Kogure K. Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects• Brain Res 1991; 553:238- 42. 16. Nishi S, Taki W, Uemura Y, et al. Ischemic tolerance due to the induction of HSP70 in a rat ischemic recirculation model. Brain Res 1993;615:281-8. 17. Tarlov IM. Spinal cord compression: mechanisms of paralysis and treatment. Springfield, IL: Charles C. Thomas, 1957;147. 18. Mutch WA, Graham MR, Halliday WC, Thiessen DB, Girling LG. Use of neuroanesthesia adjuncts (hyperventilation and mannitol administration) improves neurological outcome after thoracic aortic cross-clamping in dogs. Stroke 1993;24: 1204-11. 19. Woloszyn TT, Marini CP, Coons MS, et al. Cerebrospinal fluid drainage and steroids provide better spinal cord protection during aortic cross-clamping than does either treatment alone. Ann Thorac Surg 1990;49:78-83.
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Ann Thorac Surg 1997;63:1315-20
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Important News: New HCFA Crisis Update Section on the STS Web Site Recently the H e a l t h Care F i n a n c i n g A d m i n i s t r a t i o n (HCFA) has p r o p o s e d crippling reductions in Medicare Fee Schedules for cardiothoracic s u r g e r y - - r e d u c t i o n s that threaten our very ability to deliver health care to the vast Medicare population. The Council and m e m b e r s h i p of The Society of Thoracic Surgeons (STS) are r e s p o n d i n g in crisis mode, mobilizing efforts in every possible direction to o p p o s e this decision. As STS President George C. Kaiser e m p h a s i z e d in his recent m e m o r a n d u m , "Call to Action," "The seriousness of this p r o b l e m dictates that all cardiothoracic surgeons r e s p o n d i m m e d i a t e l y a n d decisively to this threat to disrupt the finest healthcare system ever created." A n e w " H C F A Crisis U p d a t e " section has b e e n created on the STS W e b site as a communications center to u p d a t e our m e m b e r s h i p , and any other interested parties, on these issues. This site contains important documents including details of the HCFA proposal, the STS position paper, the "Call to Action," the STS Resolution to form a PAC, and key letters to HCFA. A n on-line
© 1997 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
discussion has been started, a n d any helpful ideas or suggestions are needed. STS M e m b e r s are u r g e d to write their m e m b e r s of Congress; an STS m e m o is being p r e p a r e d suggesting key points. Robert Wilbur, STS representative in W a s h ington, DC, e m p h a s i z e s that "Letters on p a p e r are much better, and they should be individualized, not boilerplate. Follow-up on E-mail is fine after the letter, b u t letter first, please. STS m e m b e r s who know their m e m b e r of Congress should telephone. W e are a l r e a d y having positive response from t e l e p h o n e calls. Describe the basics of the p r o b l e m and ask the C o n g r e s s p e r s o n ' s office to contact me for m o r e detailed information. I have had several very good meetings with Congressional staff this w e e k following on contact from STS m e m b e r s . " The STS W e b site can help y o u - - s e e "Find Your M e m b e r of C o n g r e s s " on the HCFA Crisis U p d a t e page, at http://www.sts.org. Here is a practical chance for each of us to influence the i m m e d i a t e future of our profession.
Ann Thorac Surg 1997;63:1320 • 0003-4975/97/$17.00 PII S0003-4975(97)00406-2