Anesth Analg 2005;100:1134-1139
© 2005 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000146962.91038.15
NEUROSURGICAL ANESTHESIA
Intrathecal Ketorolac Pretreatment Reduced Spinal Cord Ischemic Injury in Rats
Ying-Chou Hsieh*
,
Wen-Yi Liang
,
Shen-Kou Tsai, and
Chih-Shung Wong
*Graduate Institute of Medical Science, National Defense Medical Center; Department of Anesthesiology, Tri-Service General Hospital and National Defense Medical Center, Neihu; and Departments of Anesthesiology and Pathology, Veterans General Hospital, Shipai, Taipei, Taiwan
Address correspondence and reprint requests to Chih-Shung Wong, MD, PhD, Department of Anesthesiology, Tri-Service General Hospital and National Defense Medical Center, Neihu, Taipei, Taiwan. Address e-mail to w82556{at}ndmctsgh.edu.tw.
 |
Abstract
|
|---|
Paraplegia caused by spinal cord ischemic injury remains a potential complication of surgical repair of thoracoabdominal aortic aneurysms. Studies suggest that cyclooxygenase (COX) contributes to ischemic neuronal damage and that COX inhibitors may reduce injury. In this study, we examined whether intrathecal pretreatment with ketorolac, a nonselective COX inhibitor, had a protective effect against ischemic spinal cord injury in rats. Rats were randomized to receive either intrathecal normal saline, ketorolac 30 µg, or ketorolac 60 µg (n = 6 rats per group) 1 h before spinal cord ischemia (intraaortic balloon occlusion combined with proximal arterial hypotension for 11 min). Another 6 rats served as the sham-operated controls. Ischemic injury was assessed by hindlimb motor function and by histopathological changes in the lumbar spinal cord at 24 h after the ischemic insult. The other 20 rats (n = 10 per group) were used in the second experiments to evaluate the safety of this drug. Survival of rats was recorded 28 days after reperfusion. Intrathecal pretreatment with 60 µg of ketorolac significantly reduced neuronal death and improved hindlimb motor function, and the long-term survival was similar to that in the control group. The results suggest that intrathecal ketorolac may be of therapeutic potential for preventing spinal cord ischemic injury during thoracoabdominal aortic surgery.
|
Introduction
|
|---|
Paraplegia, a serious complication occasionally seen after surgical repair of thoracoabdominal aortic aneurysms, has been attributed to temporary or permanent ischemia of the spinal cord caused by interruption of blood flow during aortic cross-clamping (1). The prevention of ischemic paraplegia after such surgery is a challenging issue for both anesthesiologists and surgeons. Inflammatory neurological processes have been suggested to be involved in ischemia-induced deficits. Cyclooxygenase (COX) has become a focus of attention because it is the rate-limiting enzyme in arachidonic acid metabolism and thus in the generation of prostaglandins and thromboxanes, which play important roles in the inflammatory responses (2). Two COX isoforms have been characterized. The constitutively expressed COX-1 is involved in the production of prostanoids, which are responsible for several physiological functions, whereas the inducible form, COX-2, is suggested to play a role both in normal physiological processes and in pathological processes, including inflammation. In the past decade, there has been considerable interest in the involvement of COX-2 in brain ischemia. Increased COX-2 expression has been described after transient (3) or permanent (4) middle cerebral artery occlusion and after global ischemia. Several studies have examined whether selective COX-2 inhibition helps to reduce cerebral ischemic injury (5–7). However, the relationship between COX and spinal cord ischemic injury has only been investigated in one study (2) in which the authors used a specific long-acting COX-2 inhibitor, SC-236, to improve motor function in a rabbit model of reversible spinal cord ischemia. Relatively large doses of SC-236 via intraperitoneal administration were required because of its poor penetration into the spinal cord. Most COX inhibitors are relatively insoluble in aqueous media and must be dissolved in organic solvents, such as dimethyl sulfoxide, which limits their intrathecal (IT) use.
Ketorolac, a member of the pyrrolo-pyrrole group of nonsteroidal antiinflammatory drugs (NSAIDs), is a potent antiinflammatory drug and inhibits both COX-1 and COX-2, but, in contrast to other NSAIDs, there are three crystal forms of ketorolac, all of which are water-soluble. IT administration of ketorolac at micromolar concentrations (10–5 M or 10–6 M) has a significant antinociceptive effect in rats (8,9). A phase I clinical trial (10) with a maximum dose of 2 mg of ketorolac IT has shown that it has an analgesic effect without inducing any significant neuropathological changes. The aim of the present study was to examine whether IT pretreatment with ketorolac protected rats against ischemic spinal cord injury.
|
Methods
|
|---|
The following investigations were performed using a protocol approved by the Institutional Animal Care Committee of the National Defense Medical Center, Taiwan. Male Wistar rats (400–450 g) were used. For IT drug delivery, all rats were implanted with an IT catheter at least 3 days before the induction of spinal cord ischemia (11). The rats were placed in an acrylic plastic box and anesthetized with 5% enflurane (Ohmeda, Guayama, Puerto Rico) in air. The back of the head and neck were shaved. The rats were then placed in a stereotaxic headholder with the head flexed forward and anesthesia being maintained with 2%–3% enflurane via a face mask. An incision was made in the skin from the posterior nuchal area to the top of the head, and the occipital muscles were separated from the base of the skull. A polyethylene catheter (PE-10; 9 cm) was passed through the cisternal membrane down to the region of lumbar enlargement and externalized at the back of the head. Rats showing motor impairment after IT cannulation were immediately killed.
Spinal cord ischemia was induced, as previously described (12). Anesthesia was induced in IT catheter-implanted rats in an acrylic plastic box using 5% enflurane and maintained using 2%–3% enflurane via a face mask. Body temperature, monitored with a rectal probe inserted 8 cm into the rectum, was maintained between 37.2°C and 37.5°C by a heating pad underneath the rat. The tail artery was cannulated with a 22-gauge PE catheter, which was used to monitor distal arterial pressure (DAP) and for intraarterial infusion of heparin. The left carotid artery was cannulated with a 20-gauge PE catheter (Terumo, Tokyo, Japan), which was used to monitor the proximal artery pressure (PAP) and to take blood samples. For the induction of spinal cord ischemia, the left femoral artery was exposed, and a 2F Fogarty catheter (Edwards, Irvine, CA) was passed into the thoracic aorta. The catheter tip was located at the junction with the left subclavian artery. In preliminary experiments, this level was found to correspond to a distance of 10.8–11.4 cm from the insertion site. Immediately after completion of arterial cannulation, 200 U (0.2 mL) of heparin (Leo, Ballerup, Denmark) was injected into the tail artery. The balloon was inflated with 0.05 mL of saline for 11 min to induce spinal cord ischemia. The efficiency of occlusion was documented by an immediate and sustained decrease in DAP in the tail artery. To maintain the PAP approximately 40 mm Hg during occlusion, blood from the carotid artery was allowed to flow into a 50-mL syringe filled with heparinized saline (4 U/mL of saline) positioned 54 cm above the rat. The temperature of the blood in the syringe was maintained at 37°C. After ischemia, the balloon was deflated, and the blood in the syringe was transfused back into the rat over a 60-s period. After completion of all procedures, the catheters were removed and the wounds closed. Protamine sulfate (4 mg) (Leo) was subcutaneously injected to reverse the anticoagulation effect of heparin.
In study 1, the effect of IT ketorolac (Yung-Shin Pharm (YSP), Taipei, Taiwan) on the neurological outcome and histopathological changes was examined. The rats were randomly divided into four groups as follows: (a) control-operated group C (n = 6), injected IT with saline 10 µL 1 h before the ischemia induction, (b) group K60 (n = 6), injected IT with 60 µg of ketorolac diluted in 10 µL of saline 1 h before the ischemia induction, (c) group K30 (n = 6), injected IT with 30 µg of ketorolac diluted in 10 µL of saline 1 h before the ischemia induction, and (d) sham-operated group S (n = 6), in which the balloon catheter was placed in the thoracic aorta but not inflated, and the PAP was decreased to 40 mm Hg for 11 min.
After the induction of spinal ischemia, the rats were returned to their home cages for recovery and assessed for neurological function 24 h after the operation. Motor function was quantified by assessment of ambulation using the hindlimbs placing/stepping reflex (13). Ambulation using the hindlimbs was graded as: 0 = normal (symmetrical and coordinated hind limb ambulation); 1 = toes flat beneath body when walking, but ataxia present; 2 = knuckle walking; 3 = unable to knuckle walk but some movement of the hindlimbs; and 4 = no movement of the hindlimbs. The placing/stepping reflex was assessed by dragging the dorsum of the hindpaw along the edge of a surface. This normally evokes a coordinating lifting and placing response (i.e., stepping), which was graded as follows: 0 = normal; 1 = weak; and 2 = no stepping. A motor deficit index (MDI) was calculated for each rat as the sum of both scores, with a maximum of 6 (score of 4 for ambulation and of 2 for the placing/stepping reflex). The MDI was calculated by observers unaware of the treatment used.
After evaluation of motor behavior, the rats were anesthetized with chloral hydrate (400 mg/kg, intraperitoneally) and transcardially perfused with 100 mL of heparinized saline followed by 150 mL of 4% paraformaldehyde in phosphate buffer (pH value of 7.4). The lumbar enlargement of the spinal cord at L3 or L4 was removed and postfixed overnight in the same fixative at 4°C. The specimens were embedded in paraffin, and transverse sections were (5-µm thick) cut and stained with hematoxylin and eosin. The samples were analyzed by a pathologist blinded to treatment group. The grading of acute gray matter injury was based on the percentage of abnormal or dead neurons in the ventral horns scored as: 0 = no neuronal injury or death; 1 = mild damage (<10%); 2 = moderate damage (10%–50%); and 3 = severe damage (>50%). The score for each rat was the mean for the right and left hemicords in three consecutive sections.
In study 2, the effect of IT ketorolac on long-term survival was evaluated by recording the survival rate for 28 days in the following groups: (a) saline group C (n = 10) and (b) group K60 (n = 10).
Differences in motor and histopathological scores were assessed using the nonparametric Mann-Whitney test. Fisher's exact probability test was used for statistical comparison in the survival analysis study. A P value <0.05 was considered significant.
|
Results
|
|---|
There were no differences in body weight among any of the groups. The MDI and pathological scores showed significant differences among the groups at 24 h after spinal cord ischemia (Table 1). Group S showed normal motor function, whereas group C displayed an acute flaccid paraplegia after 11 min of aortic occlusion with proximal controlled hypotension (40 mm Hg) and showed spastic paraplegia at 24 h after reperfusion. Group K60 showed near normal motor function at 24 h after spinal cord ischemia, whereas in group K30, four of six rats displayed severe spastic paraplegia, and the other two suffered slight impairment of hindlimb movement (MDIs of 4 and 5).
After 24 h, no neuronal injury or death was seen in the lumbar spinal cord of group S. Significant neuronal injury in the gray matter was seen in group C—the neurons showing features of ischemic cell death, including cytoplasmic eosinophilia with disintegration of cytoarchitecture and nuclear pyknosis. Pretreatment with 60 µg, but not 30 µg, of ketorolac resulted in a marked reduction in these histological changes (Fig. 1).
View larger version (131K):
[in this window]
[in a new window]
|
Figure 1. Photomicrographs of the lumbar enlargement of the rat spinal cord 24 h after spinal cord ischemia (X100, hematoxylin-eosin stain). Histology of the ventral gray matter in a control-operated rat (A), a sham-operated rat (B), a rat pretreated with ketorolac 30 µg (C), and a rat pretreated with ketorolac 60 µg (D).
|
|
Because pretreatment with 60 µg, but not 30 µg, of ketorolac significantly reduced the motor disturbances, we examined the therapeutic effects of 60 µg of ketorolac in a survival study. In group C (n = 10), only 2 rats survived for 28 days after ischemic injury. In group K60 (n = 10), 1 rat showed delayed paraplegia at 48 h after ischemia and died 1 day later, and 3 of the other rats died within 4 wk. The other 6 rats retained good motor function until 28 days after reperfusion. The overall survival rate at 28 days after surgery was larger in rats receiving ketorolac (60%) than in control rats (20%) (Fig. 2), but there was no significant difference between the two groups using Fisher's exact probability test for statistical comparison.
|
Discussion
|
|---|
In the present study, at 24 hours after ischemic-reperfusion, the hindlimb motor dysfunction induced by spinal cord ischemic injury was significantly attenuated in rats pretreated with a nonselective COX inhibitor (ketorolac 60 µg IT) compared with control-operated rats. Pretreatment with ketorolac 60 µg IT also prevented the histological changes in the spinal cord at 24 hours after ischemia, but the smaller dose (30 µg) had no protective effect. The neuroprotective mechanism of ketorolac (IT 60 µg) is not defined. We postulate that it may be due to its antiinflammatory effect via COX inhibition. Ketorolac is a potent, nonselective COX inhibitor with a COX-1/COX-2 activity ratio of 0.13 in rats and 0.33 in humans (14). In rodents and humans, ketorolac does not readily cross the blood-brain barrier because of its poor lipophilicity (14). In fact, ketorolac concentrations in the cerebrospinal fluid are only 0.2% of those in plasma in humans (14), suggesting that plasma concentrations would have to be increased 500-fold to obtain a therapeutic concentration of ketorolac in the cerebrospinal fluid. The mechanism of the central action of ketorolac has not been fully clarified, but the expression of COX isoforms in the rat spinal cord suggests a central COX inhibitory effect (15). The overall pharmacological profile of ketorolac is reported to favor its analgesic, rather than its antiinflammatory, activity (16); however, Jett et al. (14) stated that the association or dissociation of the analgesic and antiinflammatory effects of ketorolac depends on whether the drug is administered prophylactically or therapeutically. If ketorolac is administered prophylactically, its antiinflammatory and analgesic potencies are similar. Ischemia induces a massive inflammatory reaction, and thus, pretreatment with NSAIDs to reduce this reaction may be more efficient than after treatment. This is why we used pretreatment in the present study.
Nonselective COX inhibition had been used to reduce brain injury. A mixed COX inhibitor, BW755C, was used by Chen et al. (17) to reduce blood-brain barrier permeability and infarct volume in a rat focal brain ischemia model. Indomethacin has also been used in focal brain ischemic rats with beneficial effects (18). The reduction of harmful prostanoids via COX inhibition has become an important means of reducing brain ischemic injury. Few data are available about COX inhibition as a means of protection against spinal cord ischemic injury. In contrast to the study of Lapchak et al. (2) using the selective COX-2 inhibitor SC-236, we used the nonselective COX inhibitor ketorolac. Our results are consistent with those of Lapchak et al. (2) and suggest that the use of IT COX inhibitors may be helpful in preventing spinal cord ischemic injury.
In contrast to the proven harmful effect of COX-2 upregulation in cerebral ischemia, the role of COX-1 in neuronal ischemia is still controversial. There are reports that COX-1 expression is neither upregulated nor downregulated by ischemic brain injury and may have no effect on brain ischemia (3), that it is upregulated and may protect against cerebral ischemia (19), and that it is upregulated after focal cerebral infarctions and has harmful effects (20). Candelario-Jalil et al. (21) reported that transient global ischemia in gerbils results in a biphasic increase in COX activity, with an early increase in COX-1 activity, leading to prostaglandin E2 production and a delayed persistent increase in COX-2 activity. However, the present study focused on the behavioral and histopathological effects of ketorolac after spinal cord ischemia. Further studies are required to understand the role of COX-1 in spinal cord ischemia.
Korkmaz et al. (22) reported that chronic IT injections of ketorolac (up to 400 µg at each injection time, four times, with 5 days between injections) did not lead to any histopathological sign of injury of the spinal cord. Therefore, the maximal dosage we used (60 µg single injection) might be safe to the nervous tissue. Ketorolac did not achieve a better survival rate in rats when compared with the control group. Interruption of aortic blood flow during aortic aneurysm surgery may not only induce spinal cord ischemia, but also damage the heart, gut, and kidney. Our data showed a neuroprotective effect of IT ketorolac in reducing the incidence of paraplegia, but it may not exert a protective effect on other organs. In fact, 40% of the rats receiving ketorolac and protected from acute paraplegia died within 28 days.
Is ketorolac beneficial to the spinal cord but harmful to other organs? A single dose of IV ketorolac administration was reported not to impair renal function (23). Clinically, the most common side effects of NSAIDs are in the gastrointestinal system (24). Because our data showed the death rate was no more frequent in the ketorolac group than in the ischemic group, and the dosage used via the IT route is far less than that used by the IV route, this may not aggravate gut and other organ pathology during interruption of aortic flow. However, further studies may be required to elucidate the enigma.
In conclusion, this study demonstrated that IT administration of the nonselective COX inhibitor ketorolac, at a dose of 60 µg, attenuated the damage caused by spinal cord ischemia in rats. Motor function was improved and histopathological changes reduced compared with the control-operated group. Because spinal cord ischemia-induced paraplegia remains a serious complication of surgery involving the aorta, IT administration of ketorolac before surgery might be useful in its prevention. However, extrapolating from rats to humans in this entity must be done with caution, and testing with different doses and therapy regimens in some other animal species remains to be completed and observed by safety trials in humans before clinical use.
We thank Dr. An-Kou Chou (Department of Anesthesiology, Chang-Gung Memorial Hospital, Kaohsiung) for technical assistance and Miss Mei-Rong Lui (i-Stat Company, Taipei) for the statistical analysis.
|
Footnotes
|
|---|
Supported, in part, by a grant from the Ministry of Defense, Taiwan (DOD-93–02–08).
Accepted for publication September 17, 2004.
|
References
|
|---|
- Svensson LG. New and future approaches for spinal cord protection. Semin Thorac Cardiovasc Surg 1997;9:206–21.[Medline]
- Lapchak PA, Araujo DM, Song D, Zivin JA. Neuroprotection by the selective cyclooxygenase-2 inhibitor SC-236 results in improvements in behavioral deficits induced by reversible spinal cord ischemia. Stroke 2001;32:1220–5.[Abstract/Free Full Text]
- Nogawa S, Zhang F, Ross ME, Iadecola C. Cyclooxygenase-2 gene expression in neurons contributes to ischemic brain damage. J Neurosci 1997;17:2746–55.[Abstract/Free Full Text]
- Kinouchi H, Huang H, Arai S, et al. Induction of cyclooxygenase-2 messenger RNA after transient and permanent middle cerebral artery occlusion in rats: comparison with c-fos messenger RNA by using in situ hybridization. J Neurosurg 1999;91:1005–12.[Web of Science][Medline]
- Candelario-Jalil E, Alvarez D, Castaneda JM, et al. The highly selective cyclooxygenase-2 inhibitor DFU is highly neuroprotective when given several hours after transient cerebral ischemia in gerbils. Brain Res 2002;927:212–5.[Web of Science][Medline]
- Nakayama M, Uchimura K, Zhu RL, et al. Cyclooxygenase-2 inhibition prevents delayed death of CA1 hippocampal neurons following global ischemia. Proc Natl Acad Sci USA 1998;95:10954–9.[Abstract/Free Full Text]
- Wakita H, Tomimoto H, Akiguchi I, et al. Cyclooxygenase-2 inhibitor attenuates white matter damage in chronic cerebral ischemia. Neuroreport 1999;10:1461–5.[Web of Science][Medline]
- Conklin DR, Eisenach JC, Intrathecal ketorolac enhances antinociception from clonidine. Anesth Analg 2003;96:191–4.[Abstract/Free Full Text]
- Ma W, Du W, Eisenach JC. Intrathecal lidocaine reverses tactile allodynia caused by nerve injuries and potentiates the antiallodynic effect of the COX inhibitor ketorolac. Anesthesiology 2003;98:203–8.[Web of Science][Medline]
- Eisenach JC, Curry R, Hood DD, Yaksh TL. Phase I safety assessment of intrathecal ketorolac. Pain 2002;99:599–604.[Web of Science][Medline]
- Yaksh TL, Rudy TS. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976;17:1031–6.[Medline]
- Taira Y, Marsala M. Effect of proximal arterial perfusion pressure on function, spinal cord blood flow, and histopathological changes after increasing intervals of aortic occlusion in the rat. Stroke 1996;27:1850–8.[Abstract/Free Full Text]
- Marsala M, Yaksh TL. Transient spinal ischemia in the rat: characterization of behavioral and histopathological consequences as a function of the duration of aortic occlusion. J Cereb Blood Flow Metab 1994;14:526–35.[Web of Science][Medline]
- Jett MF, Ramesha CS, Brown CD, et al. Characterization of the analgesic and anti-inflammatory activities of ketorolac and its enantiomers in the rat, J Pharmacol Exp Ther 1999;288:1288–97.[Abstract/Free Full Text]
- Beiche F, Brune K, Geisslinger G, Goppelt-Struebe M. Expression of cyclooxygenase isoforms in the rat spinal cord and their regulation during adjuvant-induced arthritis. Inflamm Res 1998;47:482–7.[Web of Science][Medline]
- Rooks WH. The pharmacologic activity of ketorolac tromethamine. Pharmacotherapy 1990;10:30S–2.[Medline]
- Chen J, Weinstein PR, Graham SH. Attenuation of postischemic brain hypoperfusion and reperfusion injury by the cyclooxygenase-lipoxygenase inhibitor BW755C. J Neurosurg 1995;83:99–104.[Web of Science][Medline]
- Buccellati C, Folco GC, Sala A, et al. Inhibition of prostanoid synthesis protects against neuronal damage induced by focal ischemia in rat brain. Neurosci Lett 1998;257:123–6.[Web of Science][Medline]
- Lin H, Lin TN, Cheung WM, et al. Cyclooxygenase-1 and bicistronic cyclooxygenase-1/prostacyclin synthase gene transfer protect against ischemic cerebral infarction. Circulation 2002;105:1962–9.[Abstract/Free Full Text]
- Schwab JM, Nguyen TD, Postler E, et al. Selective accumulation of cyclooxygenase-1-expressing microglial cells/macrophages in lesions of human focal cerebral ischemia. Acta Neuropathol (Berl) 2000;99:609–14.[Medline]
- Candelario-Jalil E, Conzalez-Falcon A, Garcia-Cabrera M, et al. Assessment of the relative contribution of COX-1 and COX-2 isoforms to ischemia-induced oxidative damage and neurodegeneration following global cerebral ischemia. J Neurochem 2003;86:545–55.[Web of Science][Medline]
- Korkmaz HA, Maltepe F, Erbayraktar S, et al. Antinociceptive and neurotoxicologic screening of chronic intrathecal administration of ketorolac tromethamine in the rat. Anesth Analg 2004;98:148–52.[Abstract/Free Full Text]
- Jaquenod M, Ronnhedh C, Cousins MJ, et al. Factors influencing ketorolac-associated perioperative renal dysfunction. Anesth Analg 1998;86:1090–7.[Abstract]
- Wallace JL, McKnight W, Reuter BK, Vergnolle N. NSAID-induced gastric damage in rats: requirement for inhibition of both cyclooxygenase 1 and 2. Gastroenterology 2000;119:706–14.[Web of Science][Medline]
Top
Abstract
Introduction
