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Eur J Cardiothorac Surg 2004;26:351-358
© 2004 Elsevier Science NL
a Department of Cardiothoracic Surgery, Guy's Hospital, St Thomas Street, London SE1 9RT, UK
b Department of Cardiovascular Surgery, Institut Mutualiste Montsouris, Paris, France
c Bristol Heart Center, Bristol University, Bristol, UK
Received 27 October 2003; received in revised form 4 March 2004; accepted 8 March 2004.
* Corresponding author. Tel.: +44-207-188-1038; fax: +44-207-955-4858
e-mail: loic.lang-lazdunski{at}gstt.nhs.uk
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Abstract |
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 Top Abstract 1. IntroductionAppendix A. Conference...References |
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Key Words: Thoracic aortic surgery • Spinal cord ischemia • Open distal anastomosis
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1. Introduction |
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TopAbstract1. IntroductionAppendix A. Conference...References |
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In 1992, Dr Cooley has advocated a new technique ‘open distal anastomosis’ for the repair of descending thoracic or thoracoabdominal aortic aneurysms [9]. This technique consisted in using only a single proximal clamp on the aorta and performing an open distal anastomosis. No attempt was made to control back-bleeding from the distal aorta or segmental vessels during implantation of the tube-graft. Dr Cooley argued that distal exsanguinations attenuate the rise in CSF pressure seen with proximal aortic cross-clamping and improve thereby spinal cord perfusion. He has recently reported his results in 132 consecutive patients sustaining thoracic or thoracoabdominal aortic graft-replacement, with early mortality being 12.9% and spinal cord ischemia occurring in 8.3% [10]. Dr Cooley stated that this technique yielded an acceptable complication rate whenever cross-clamp time did not exceed 30 min [10].
Some have argued that segmental vessels back-bleeding may reduce spinal cord perfusion and increase spinal cord ischemic jeopardy [11]. Others have emphasized the deleterious impact of reducing distal aortic pressure on the incidence of neurological deficits [5,12].
Because little is known about the safety of performing the distal anastomosis ‘open’, we have developed a model of open distal anastomosis in the rat and have examined the impact of this technique on lumbar spinal cord blood flow, neurological outcome and spinal cord histopathology.
2. Materials and methods
2.1. Animal care and surgical technique
Sprague–Dawley male rats weighing 350–400 g were used in this study. All animals were allowed free access to laboratory chow and tap water in day/night regulated quarters at 25 °C. Animal care and experiments complied with ‘The Principles of Laboratory Animal Care’ (Guide for the Care and Use of Laboratory Animals, NIH publication 86-23, 1985) and was approved by the local Animal Studies Committee.
Anesthesia was induced in a chamber containing 3% halothane and was maintained by inhalation through a facial mask of 1.5% halothane driven by oxygen 2 l/min. Rectal temperature was continuously monitored with a flexible probe inserted 3 cm into the rectum, and supported by a thermal pad and a heating lamp. Body temperature was maintained at 37±0.5 °C during the surgical procedure. The rats were placed in the supine position and a longitudinal incision was made on the left groin. The left femoral artery was encircled with silk sutures. The tail artery was catheterized with a 22 F catheter for monitoring of distal arterial blood pressure (DABP) and for collecting blood samples. Then, rats received 100 U of heparin (0.1 ml) injected into the tail artery. A right groin incision was made and the right femoral artery was encircled with silk sutures. The proximal descending thoracic aorta was transiently occluded by inflating the balloon of a 2 F Fogarty catheter (model 120602F, Baxter Healthcare) with 0.1 ml of sterile water after its insertion through the left femoral artery and its advancement 11 cm cephalad from the arteriotomy site. Previous studies have indicated that this length of catheter advancement resulted in positioning of the catheter balloon in the proximal descending thoracic aorta just distal to the origin of the left subclavian artery [13]. The efficiency of aortic occlusion was confirmed by monitoring DABP. After aortic occlusion for 15 min, the balloon was deflated and the Fogarty catheter was withdrawn. The left femoral artery was ligated.
Rats were separated into three groups:
Stabilization of the DABP was monitored for 20 min. Then, catheters were removed and the wounds were closed after povidone iodine irrigation.
Animals were allowed to recover in a plastic box at 28 °C for 3 h, and were placed in their cages. Credé's manoeuver was used twice daily to empty the urinary bladder of paraplegic animals.
2.2. Measurement of lumbar spinal cord blood flow (SCBFl)
Five animals in both the control and open distal groups had measurement of SCBFl during aortic occlusion. Rats were placed in the prone position and the L2 vertebra was exposed by midline incision and blunt dissection. A small hole was then drilled on the right side of the lamina overlying the L4 and L5 spinal segments. We used a qualitative real-time measure of SCBFl by laser-Doppler flowmetry. The probe of a 0.8-mm fiberoptic extension (PF2B, Perimed) was placed into the epidural space and affixed with glue. SCBF was then continuously monitored before, during, and after aortic occlusion. At the end of the procedure, the tip of the fiberoptic extension was cut and left in place. The wound was irrigated with povidone iodine and closed in layers.
2.3. Physiological parameters
DABP, arterial blood gases and pH were measured throughout the procedure. A Mac Intosh computer and a Mac LAB/8 System equipped with an ETH-400 transducer amplifier were used for continuous acquisition and online analysis of data. Arterial blood gases and pH were measured 10 min before aortic occlusion and 10 min after the onset of reperfusion in samples obtained from the tail artery, using a blood gas/pH analyzer (Corning 178, Ciba-Corning Diagnostics). Blood glucose levels were measured in all animals before aortic occlusion.
2.4. Evaluation of neurobehavioral outcome
Serial assessments of motor function in the hindlimbs of all rats were performed at 6, 24 and 48 h of reperfusion, using a system described by Kanellopoulos et al. [13]. A motor deficit score (MDS) was given to each rat according to the following criteria:
Rats with MDS
3 were considered paraplegic, whereas rats with MDS<3 were considered non-paraplegic, in this study.
2.5. Euthanasia
Animals were sacrificed after 48 h of reperfusion. Animals were anesthetized with 3% halothane and transcardially perfused with 100 ml of 0.9% saline solution at 4 °C followed by 50 ml of fresh 4% paraformaldehyde at 4 °C. Spinal cords were quickly harvested, and placed in fresh 4% paraformaldehyde at 4 °C for 48 h.
2.6. Histopathology
Spinal cords were removed from 4% paraformaldehyde after 48 h fixation. Specimens were dehydrated in alcohol 95% for 30 min, followed by four changes of 100% alcohol for 1 h each and five changes of toluene for 1 h each under vacuum at 37 °C. Spinal cords were infiltrated with paraffin and embedded in paraffin at 57 °C under vacuum and pressure. Transverse sections were made on a microtome (Leica, Rueil Malmaison, France). Ten-micrometer sections were obtained through the lumbar cord. Sections were stained with hematoxylin and eosin (H&E) and Luxol fast blue staining method and were examined under the light microscope. All rats had their spinal cord examined. Five representative sections taken from the L1 to L5 segments were examined in each animal. The neuropathologist who performed the examination was blinded to the experimental conditions. He quoted the damage in gray matter and counted the motor neurons in each section. The Rexed's classification was used to depict the locations of damaged neurons in gray matter [14].
2.7. Immunohistochemistry for MAP-2
The cytoskeletal protein MAP-2 is involved in maintaining neuronal structural integrity. It is extremely sensible to ischemia and immunoreactivity for MAP-2 has been demonstrated to be a sensitive, accurate and early marker of spinal cord injury following ischemia [15].
Sections were immersed in 0.3% H2O2 for 10 min, blocked with 5% goat serum and 3% Triton for 1 h at room temperature, followed by a rinse in PBS. The sections were incubated with the primary antibody overnight. The antiserum used for the study of cytoskeletal protein expression was a monoclonal mouse anti-MAP-2 (Clone HM-2, Sigma, diluted 1:500). After the primary incubation and three rinses in PBS, sections were incubated in biotinylated horse anti-mouse IgG (diluted 1:100, Vector Labs.) for 3 h. MAP-2 expression was visualized by 3'-diaminobenzidine and nickel chloride (DAB-Ni) staining using the vectastain ABC kit (Vector Labs.). All sections were washed a final time in PBS and distilled water and then mounted with glycerol. Sections were examined under the light microscope.
2.8. Statistical analysis
Spinal cord blood flow and motor neurons counts were compared using analysis of variance. Fisher's exact test was used to compare neurological scores and mortality across the groups. Blood gases and DABP during reperfusion were compared using analysis of covariance, adjusted for baseline measurements. The DABP measurements at the end of aortic occlusion and during reperfusion were also compared using a mixed regression model. The mixed model allows the data for the two time points to be analyzed together, whilst recognizing that repeated measurements from the same animal are not independent, but correlated. Alternative correlation structures were explored and the structure that gave the lowest value for Akaike's Information criteria was chosen. This analysis was adjusted for both baseline DABP and DABP measured just after aortic occlusion.
3. Results
3.1. Physiological parameters
The preocclusion arterial blood gases were within normal range for all groups and the differences between the three groups were not statistically significant. Arterial blood gases taken 10 min after the balloon deflation demonstrated severe metabolic acidosis, particularly in the open distal group (Table 1). pH after 10 min of reperfusion was different in all three groups (P<0.001), although the difference between control rats and those in the open distal group was very small. PaCO2 and standard bicarbonate also differed significantly across the groups (P=0.03 and <0.001, respectively). PaO2 did not differ across the groups (P=0.27) (Table 4).
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Balloon inflation resulted in a rapid decrease of the DABP in all rats. The maximal DABP measured during the period of aortic occlusion and measured after 10 min of reperfusion were significantly lower in the open distal group compared with the control group (Table 2, P<0.001).
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3.2. Spinal cord blood flow
Stepwise decrease in DABP after aortic occlusion caused a dramatic reduction of SCBF that reached 5% of baseline just after balloon inflation, and was 10.6±0.9% before balloon deflation in the control group and 6±0.7% of baseline in the open distal group. SCBF returned to baseline within 5 min following release of aortic occlusion. SCBF measured during aortic occlusion varied significantly between the control rats and those in the open distal group (P<0.001, mean difference was –4.6, with 95% confidence interval –5.8 to –3.4).
3.3. Neurobehavioral outcome and mortality
All rats recovered from anesthesia. No rat died in the sham-operation group, whereas 48-h mortality was 6.6% (1/15) in the control group and 46.6% (7/15) in the open distal group (P=0.02). All rats that died in the open distal group had bowel infarct and hemorrhagic dilated bladder at autopsy. In addition, focal renal infarcts could be observed in five of them (33%).
No rat recovered from anesthesia with paraplegia in the sham-operation and control groups, whereas all rats in the open distal group recovered from anesthesia with flaccid paraplegia. No rat in the sham-operation group and control group became paraplegic, whereas 13 rats (87.7%) in the open distal group suffered severe and definitive paraplegia and two (13.3%) suffered moderate neurological deficit (MDS=2) (P<0.001).
3.4. Histopathology
H&E and luxol fast blue staining method was used to analyze neuronal cell death in gray matter after 15 min of ischemia and 48 h of reperfusion. The extent of ischemic damage was grossly proportional to the MDS score, as previously reported by others [13]. Ischemic damage was observed mainly in gray matter, which contained typically necrotic neurons with eosinophilic cytoplasm (‘red neurons’) and loss of cytoplasmic structures (‘ghost neurons’), but also neurons demonstrating apoptotic features.
Sham-operated rats had normal spinal cords. Thirteen rats (87.7%) in the open distal group had very severe injury in gray matter with extended infarcts involving laminae 3–10 and containing neutrophils and mononuclear phagocytes infiltrates. Only very few motor neurons appeared to have survived the ischemia/reperfusion insult. In some cases, white matter surrounding gray matter was damaged also. Two rats (13.3%) in the open distal group had moderate injury involving mostly central gray matter (laminae 3–7) associated with relative preservation of laminae 8 and 9. All control rats had virtually normal spinal cords. The number of intact motor neurons per section is depicted in Table 3. It was significantly lower in rats sustaining partial exsanguinations (control 24±1 vs. open distal, 7±2, P<0.001).
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The most critical factor determining recovery of neurological function after transient spinal cord ischemia appears to be the magnitude of the reduction of spinal cord blood flow during aortic occlusion [4,5,12]. During aortic cross-clamping, the spinal cord functions in a low-flow state [4,5,12]. Since human spinal cord has an heterosegmental blood supply, the residual blood flow must arise from collateral vascularization. Maintenance of that collateral perfusion appears critical in determining survival of spinal cord neurons during extended cross-clamp times [12].
Another critical factor is CSF pressure. Blaisdell and Cooley have demonstrated that proximal thoracic aortic cross-clamping resulted in an immediate rise of CSF pressure [20]. Spinal cord perfusion depends on CSF pressure: thus, lowering CSF pressure may theoretically improve spinal cord blood flow (spinal cord perfusion pressure=spinal artery pressure–CSF pressure) [20,21]. Studies in dogs and humans have confirmed that CSF drainage has a protective effect against spinal cord ischemic injury during replacement of the descending thoracic and thoracoabdominal aorta [20–22]. Phlebotomy can reverse the increase in CSF pressure induced by proximal thoracic aortic cross-clamping by decreasing central venous pressure and by modifying the blood volume redistribution and intracranial hypervolemia [5,23]. However, phlebotomy per se does not improve neurological outcome following thoracic aortic cross-clamping, although it does not compromise spinal cord perfusion pressure [5,23].
The safety of exsanguinations through the distal aorta remains to be demonstrated. Although exsanguinations has the potential to decrease central venous pressure and subsequently CSF pressure, there is yet no proof that free drainage of blood through the distal aorta and segmental vessels can increase, or even preserve, spinal cord perfusion. Instead of that, this single-clamp technique may result in a real ‘steal phenomenon’ by allowing free drainage of blood away from the high-resistance spinal cord collaterals [11].
The present study demonstrates that equilibrating distal aortic pressure with atmospheric pressure results in a dramatic reduction in lumbar spinal cord blood flow. Critical ischemic time has been reported 30–40 min in the present rat model, when avoiding free drainage of distal aortic blood during occlusion of the thoracic aorta [12]. Of concern is the fact that the critical ischemic time is unpredictable in man, but is also grossly 30 min [5]. The present study demonstrates that allowing free drainage of distal aortic blood during occlusion of the thoracic aorta significantly reduces the critical ischemic time to less than 15 min in rats. Histopathologic evaluation of spinal cord specimens also strongly suggests that the depth of ischemia was much more severe in rats undergoing exsanguinations through the femoral artery. Extensive neuronal necrosis and apoptosis was observed throughout gray matter and specially in the laminae containing motor neurons. If one considers that use of the open distal anastomosis technique shorten critical spinal cord ischemic time by twofold in the rat, this would result in a safe cross-clamping time of only 15 min in man.
We decided to monitor epidural blood flow by laser Doppler flowmetry. This technique has been largely validated [12,24] and excellent correlation was found between regional spinal cord blood flow and epidural blood flow over a physiological range of PaCO2 [24]. Moreover, the rates of change in regional spinal cord blood flow and epidural blood flow were almost the same in response to both PaCO2 and blood pressure variations [25]. However, it as been argued that this technique of measurement probably reflected blood flow in dorsal white matter tracts, where blood is supplied by the posterior spinal arterial system [24]. Hence, SCBF recording might be overestimated if one considers deeper spinal cord structures such as gray matter that are mostly supplied by the anterior spinal arterial system. Therefore, we can anticipate that lumbar spinal cord blood flow in the central gray matter of rats assigned to the open distal group decreased to values less than 6% during thoracic aortic occlusion.
Another factor to consider is the development of visceral ischemia in the open distal group. Thus, both renal and bowel ischemia can result in prohibitive morbidity and mortality after repair of thoracoabdominal aortic aneurysms [3]. Mortality was dramatically increased in the open distal group (46.6 vs. 6.6%, P<0.01) and this may be attributed at least in part to spinal cord ischemic injury, but also to renal and bowel ischemic injury. These results compares with those in humans. Thus, mortality exceeding 50% has been reported in patients suffering visceral ischemia after repair of thoracoabdominal aortic aneurysms [11]. As spinal cord blood flow, blood flow through other organs below the proximal aortic clamp depends mainly on distal aortic pressure. Significant reduction in distal aortic pressure results in reduction of blood flow through the kidneys and bowel with subsequent ischemia, depending on the duration of blood flow reduction [11]. Metabolic acidosis may result from bowel and kidney hypoperfusion [11]. In the present study, metabolic acidosis was significantly more severe in rats undergoing exsanguinations and it cannot be excluded that acidosis had contributed to worsen spinal cord ischemic injury in the open distal group [26].
Although further studies in larger animal species are warranted to confirm those data, we suggest that the open distal anastomosis technique is used with caution during repair of the descending thoracic or thoracoabdominal aorta in man.
5. Conclusion
Allowing free drainage of distal aortic blood during thoracic aortic cross-clamping resulted in a dramatic reduction in spinal cord blood flow in rat. It also resulted in a dramatic increase in visceral ischemia, paraplegia and mortality rates. Considering our data, we estimate that use of the open distal anastomosis technique may be dangerous and should therefore be avoided whenever possible during repair of the descending thoracic or thoracoabdominal aorta in man.
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Footnotes |
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Appendix A. Conference discussion |
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TopAbstract1. IntroductionAppendix A. Conference...References |
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Mr Lang-Lazdunski: We did record distal arterial blood pressure with a tail artery catheter and a Maclab® system, and we did record spinal cord blood flow with laser Doppler flowmetry. We used a small laser Doppler from Perimed®. This was done by placing the tip of a fiberoptic probe in the epidural space through a small hole in the lamina of the L1 vertebra, and blood flow was expressed as a percentage of baseline value.
Dr S. Takamoto (Tokyo, Japan): You mentioned about temperature first but you didn't mention about temperature during this experiment. Did you cool the rat model during clamping?
Mr Lang-Lazdunski: No. Temperature was supported by a heating lamp and a thermal pad and all rats were operated in strict normothermia. The goal was to have all rats with a temperature between 37 and 37.5°C.
Dr Takamoto: And the other question is in the human operation, we could connect the intercostal arteries before we do the distal open anastomosis, so that probably we could preserve the flow to the spinal cord before distal open anastomosis, and also in the human cases we could cool the patient. So that the situation is different in this experiment from the human operation.
Mr Lang-Lazdunski: No, the situation is not different, because in that model, the critical ischemic time for the spinal cord has been proven to be between 30 and 40 min, and this is grossly the same in man. This study demonstrates that performing the distal anastomosis open decreased this critical ischemic time to less than 15 min, and in 15 min time you cannot perform the proximal anastomosis and reimplant the intercostal or critical lumbar arteries, and I guess even Dr Cooley cannot complete the full operation in 15 min.
Mr R. Ascione (Bristol, UK): In reality, what we often see in patients needing an operation of the thoracic aorta is a presentation with thoracoabdominal aneurysm developing over several months, if not years. The chronic nature of this disease may lead to the formation of new collaterals, so that when doing the operation, one may with impunity ligate some intercostals, as also suggested recently by positioning of intravascular stents. Of course, your model could not take account of this. So do you think that, in reality, the tolerance to ischemia might be better due to the formation of new collaterals?
Mr Lang-Lazdunski: I think it is very important to occlude as much nonfunctional intercostal arteries with small plastic plugs, and that had been recommended by Dr. Borst some years ago. But the important thing is to clamp the distal aorta, because you can have collateral blood supply from very low lumbar arteries, and especially to the cauda equina, that can perfuse retrogradely the anterior spinal axis. So that is very important.
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References |
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TopAbstract1. IntroductionAppendix A. Conference...References |
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  Y. Kawanishi, K. Okada, H. Tanaka, T. Yamashita, K. Nakagiri, and Y. Okita The adverse effect of back-bleeding from lumbar arteries on spinal cord pathophysiology in a rabbit model J. Thorac. Cardiovasc. Surg., June 1, 2007; 133(6): 1553 - 1558. [Abstract] [Full Text] [PDF]
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