Ann Thorac Surg 2000;70:1496-1500
© 2000 The Society of Thoracic Surgeons
Original articles: cardiovascular
Exogenous aspartate neurotoxicity in the spinal cord under metabolic stress in vivo
Yasunori Cho, MDa,
Toshihiko Ueda, MDa,
Atsuo Mori, MDa,
Tsukasa Nakamichi, MDa,
Hideyuki Shimizu, MDa,
Yoshito Inoue, MDa,
Shiaki Kawada, MDa
a Department of Cardiovascular Surgery, Keio University, Tokyo, Japan
Address reprints requests to Dr Cho, Department of Cardiovascular Surgery, Keio University, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan
e-mail: noricho{at}aol.com
 |
Abstract
|
|---|
Background. Considerable evidence exists that neurotoxicity of excitatory amino acids is related to the neuronal injury, including paraplegia. However, little is known about aspartate neurotoxicity in the spinal cord in vivo. We evaluated the detrimental effects of exogenous aspartate on spinal cord neurons under metabolic stress.
Methods. New Zealand white rabbits underwent an infrarenal aortic isolation. Group A animals (n = 7) received segmental aspartate 50 mmol/L) infusion for 10 minutes. Group B animals (n = 7) received saline as a negative control. Group C animals (n = 5) received segmental aspartate 100 mmol/L) infusion for 5 minutes. Group D animals (n = 7) were pretreated with segmental infusion of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptan-5,10-imine (MK-801) (6 mg/kg), a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist for 1 minute, followed by segmental infusion of aspartate (50 mmol/L) for 9 minutes. Group E animals (n = 7) received vehicle only, followed by aspartate (50 mmol/L) infusion as a control of group D. Neurologic status was assessed at 12, 24, and 48 hours after operation using the Tarlov score.
Results. Group A animals exhibited paraplegia or paraparesis with marked neuronal necrosis. Group B and C animals recovered fully. Group D animals showed significantly better neurologic function (p = 0.0007) compared with group E animals that exhibited paraplegia or paraparesis.
Conclusions. Exogenous aspartate can have detrimental effects on spinal cord neurons under metabolic stress. This model may be useful in assaying neuronal injury mediated by NMDA receptor in vivo.
|
Introduction
|
|---|
Paraplegia is a devastating complication associated with the repair of thoracic and thoracoabdominal aorta. Growing evidence has revealed that excitatory amino acids (EAAs), mainly glutamate and aspartate, contribute to spinal cord injury under metabolic stress, such as ischemia, hypoxia, hypoglycemia, and trauma. EEAs are major excitatory neurotransmitters in the central nervous system including the spinal cord. However, it has been difficult to indicate EEAs neurotoxicity in vivo [1]. The defense mechanism against the neurotoxicity is assumed to be caused by the activity of rapid, high affinity reuptake systems, present in presynaptic nerve terminal and astrocyte, that maintain extracellular EAAs concentration within nontoxic level [2](Figure 1).
View larger version (30K):
[in this window]
[in a new window]
|
Fig 1. Schematic illustration of the synaptic components that contribute to excitatory amino acid neurotoxicity. (1) Release of excitatory amino acids. (2) Excitatory transmission. (3) Regulation of extracellular excitatory amino acid concentrations by rapid high affinity reuptake systems. (4) Mechanisms that lead to excitatory amino acid receptor mediated neuronal injury.
|
|
We developed the segmental glutamate infusion model and showed the detrimental effects of exogenous glutamate on spinal cord neurons during 5 minutes of ischemia in vivo [3]. We performed infrarenal aortic clamping to reduce the efficiency of the reuptake systems that are highly dependent on the adenosine triphosphate energy store. This procedure caused the surge of glutamate to reach toxic level that led to neuronal injury.
Choi and colleagues [4] were the first to demonstrate aspartate neurotoxicity in cortical cell culture in vitro. However, neurotoxicity of exogenous aspartate in the spinal cord is yet to be demonstrated in vivo, and the role and impact of aspartate in spinal cord neuronal injury has not been well elucidated. We undertook this study to evaluate neurotoxicity of exogenously administrated aspartate under metabolic stress in vivo. In addition, we examined the protective effects of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptan-5,10-imine (MK-801), a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor, against the spinal cord injury.
|
Material and methods
|
|---|
Experimental model
Male New Zealand white rabbits weighing 3.0 to 3.5 kg were anesthetized with 1.5% halothane and 98.5% oxygen. The animals were placed in the supine position, maintaining spontaneous breathing without endotracheal intubation or mechanical ventilation. Central ear arterial pressure and rectal temperature were continuously monitored. A warming blanket was placed underneath each animal to maintain normal body temperature. Each animal underwent an operation in sterilized conditions using a median laparotomy. The abdominal aorta was dissected just inferior to the left renal vein and above the bifurcation. Heparin sulfate (60 U/kg intravenous bolus) was used for systemic anticoagulation, and a 20-G catheter was introduced over a guidewire from the right femoral artery to the abdominal aorta. The tip of this catheter, which could be identified through the aorta, was placed at 5 mm above the bifurcation.
Group A rabbits (n = 7) received segmental infusion of 50 mmol/L aspartate solution, which was adjusted to physiologic osmolarity with sodium chloride (NaCl) and distilled water through a femoral arterial catheter. At the beginning of the infusion the abdominal aorta was clamped with a vascular clip immediately inferior to the left renal vein, and the femoral arterial catheter was snared around the bifurcation. The posterior mesenteric artery was also clamped with a vascular clip. The aspartate solution was incubated at 39°C and was infused into the isolated segment at a rate of 2 ml/min for 10 minutes. At the end of the infusion, the clamps were released and the snare around the bifurcation loosened. The infusion catheter was removed and the abdomen was closed in two layers. In group B (n = 7), saline solution alone, incubated at 39°C, was infused at the same rate and in the same manner. Group C animals (n = 5) received segmental infusion of 100 mmol/L aspartate at the rate of 2 ml/min for 5 minutes.
In addition to these groups, three animals were treated identical to group A animals (aspartate sham) but did not undergo aortic clamping.
Pharmacologic study
MK-801 compound was obtained from Merck & Co, Inc (Rahway, NJ). The compound was dissolved in 0.9% sodium chloride (NaCl) solution at a concentration of 6 mg/ml and incubated at 39°C. Group D rabbits (n = 7) were pretreated with segmental infusion of MK-801 (6 mg/kg) through a femoral catheter for 1 minute, followed by segmental infusion of 50 mmol/L aspartate at a rate of 2 ml/min for 9 minutes. Group E rabbits (n = 7) received only vehicles of MK-801 treatment, followed by aspartate (50 mmol/L) segmental infusion in the same manner as group D. In addition, three animals (MK-801 sham) were also treated identical to group D animals without aortic clamping.
Neurologic and histopathologic evaluations
Hind limb function was evaluated at 12, 24, and 48 hours after operation according to the modified Tarlov score (5 = normal hop, 4 = weak hop, 3 = sits alone, 2 = sits with assistance, 1 = slight movement, 0 = no movement). After 48 hours, animals were reanesthetized and sacrificed with an overdose of intravenous pentobarbital. The spinal cord was fixed by perfusion of 10% formaldehyde solution through a femoral arterial catheter in the same manner as the first operation, followed by immersion fixation for 2 weeks. Cross-sections from the lower thoracic through the sacral cord were stained with hematoxylin and eosin and Luxol-fast blue, and histopathologic assessment using light microscopy was done by a neuropathologist blinded to the experimental group.
Statistical analysis
We used the Mann-Whitney U test to compare the postoperative neurologic status between the groups. Other parameters were compared between the two groups by analysis of variance for repeated measures or Student’s t test where appropriate. A p value of 0.05 was taken as the level of significance.
Animal care
All experimental animals received human care and treatment according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Publication 85–23; revised 1985).
|
Results
|
|---|
In group A, 6 of the 7 rabbits exhibited paraplegia (Tarlov 0) at 12, 24, and 48 hours after operation. The other rabbit showed severe motor dysfunction (Tarlov 2) that developed delayed paraparesis at 48 hours after operation (Table 1). Group B and C animals remained neurologically intact (Tarlov 5) at all the time points. Group A rabbits had significantly worse neurologic score compared with group B (p = 0.0005).
In group D, all rabbits could not be evaluated because of the anesthetic effect of MK-801 that lasted for more than 12 hours after the operation. Four of the 7 rabbits recovered fully (Tarlov 5) at 48 hours and the other 3 rabbits exhibited mild disturbance of motor function (Tarlov 4) at 48 hours (Table 1). In group E, 5 of the 7 rabbits exhibited paraplegia at all time points (Tarlov 0), and the other 2 rabbits showed delayed paraplegia at 48 hours (Table 1). Group D rabbits had a significantly better neurologic score compared with group E (p = 0.0007).
Histopathology revealed neuronal injury in group A. Sections exhibited severe and extensive gray matter necrosis with prominent vacuolation, numerous degenerated neurons and vascular necrosis in both anterior and dorsal horns of the lower thoracic and sacral cord. Degenerated neurons were shrunken and lightly eosinophilic and their nuclei were irregularly shrunken and stained hyperchromatic (Fig 2). On the contrary, the histology of group B and C animals remained normal (Fig 3). In group D, 4 of the 7 rabbits demonstrated normal histology (Fig 4). The other rabbits that exhibited disturbance of motor functions showed minimal neuronal change with shrinkage. However, these very focal lesions were found in one of several sections of the sacral cord. Group E animals exhibited severe and extensive gray matter necrosis as in group A .
View larger version (124K):
[in this window]
[in a new window]
|
Fig 2. Representative photomicrographs of histologic sections of the spinal cord demonstrating severe and extensive gray matter necrosis. (A) Section from group A, x 26, stained with hematoxylin-eosin and Luxol-fast blue. (B) Section from group A, x 130, stained with hematoxylin-eosin and Luxol-fast blue.
|
|
View larger version (131K):
[in this window]
[in a new window]
|
Fig 3. Representative photomicrographs of histologic sections of the spinal cord showing normal histology. (A) Section from group B, x 26, stained with hematoxylin-eosin and Luxol-fast blue. (B) Section from group B, x 130, stained with hematoxylin-eosin and Luxol-fast blue.
|
|
View larger version (125K):
[in this window]
[in a new window]
|
Fig 4. Representative photomicrographs of histologic sections of the spinal cord. Note that the spinal cord neurons were protected by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptan-5,10-imine (MK-801) against aspartate infusion. (A) Section from group D, x 26, stained with hematoxylin-eosin and Luxol-fast blue. (B) Section from group D, x 130, stained with hematoxylin-eosin and Luxol-fast blue.
|
|
The aspartate sham and MK-801 sham animals exhibited full recovery. Sections from those demonstrated normal histology.
Rectal temperatures, before the infrarenal aortic clamp, were similar in group A and group B, and group D and group E, respectively. There were no significant differences between the groups in mean systemic blood pressure and heart rate during the experiment.
|
Comment
|
|---|
We previously reported that exogenous 50 mmol/L glutamate administered during 5 minutes of ischemia showed neurotoxicity in the spinal cord [3]. In the present study, we showed that 50 mmol/L aspartate administered regionally during 10 minutes of ischemia caused neuronal injury mediated by NMDA receptor in the spinal cord.
In the aspartate sham group, the given dose of aspartate was cleared by the neurons without toxic effect. However, segmental infusion of 50 mmol/L aspartate solution might increase the concentration to a level exceeding the pathophysiologic range of aspartate release in the spinal cord. In the preliminary study, we failed to show aspartate neurotoxicity in a lower dose; we estimate that it is probably because of the blood-brain barrier that lowers the concentration of aspartate at the level of synaptic cleft. Also, it should be noted that almost infused aspartate might be washed out by reperfusion. Thus, 50 mmol/L aspartate was required to initiate intracellular vicious cycle as short as 10 minutes by aspartate neurotoxicity alone.
Group C animals (100 mmol/L aspartate with 5 minutes of isolation group) could not induce neuronal injury even though 100 mmol/L aspartate was administered. As to the clamping time, more prolonged ischemia is required to demonstrate exogenous aspartate neurotoxicity comparing with glutamate in rabbit spinal cord neurons. There are probably three factors that are responsible for the difference between aspartate and glutamate infusion: (1) Glutamate can activate both NMDA and nonNMDA receptors in postsynaptic neuronal membrane, whereas aspartate can activate only NMDA receptors [5]. (2) It was demonstrated that spinal cord neurons were less vulnerable to NMDA receptor agonists and more vulnerable to nonNMDA receptor agonists than cerebral neurons in rodents [6]. This is probably because of the lower density of NMDA receptors in the spinal cord than in the brain. (3) Another factor is the characteristic energy-dependent reuptake system. A sodium-dependent reuptake system present in both astrocyte and presynaptic nerve terminals is believed to account for the majority of transport of aspartate and glutamate at synapses. Anderson and colleagues [2] demonstrated that a sodium-dependent reuptake system has higher affinity for aspartate than glutamate. According to the reports using stereotactic microdialysis, the glutamate level was elevated during ischemia, however, a significant elevation of the aspartate level was detected only in later phases of ischemia [7, 8]. These studies suggest that reuptake activity of aspartate is more rapid and efficient than that of glutamate. It is further supported by the clinical evidence, reported by Brock and colleagues [9] that aspartate levels in cerebrospinal fluid were increased only during late reperfusion, whereas glutamate levels were especially increased during aortic clamping and late reperfusion among the patients who had clinically neurologic deficits in lower extremity after thoracoabdominal aortic aneurysm repair.
The higher concentration of EEAs in synaptic cleft induces neuronal injury in a manner of the activation of EEAs receptors, namely, NMDA and nonNMDA receptors. The activation of NMDA receptors causes calcium ion influx and is followed by activation of several calcium-dependent enzymes, including protein kinase C, phospholipase A2, nitric oxide synthase, and other proteases, leading to late neuronal degeneration. On the other hand, the activation of nonNMDA receptors mainly cause sodium ion influx and induce acute cell swelling (Figure 1). Recently, apoptosis mediated by free radicals, has been implicated in the delayed onset neuronal injury [10, 11]. In the present study, 2 rabbits from groups A and E were reported to exhibit delayed paraplegia or paraparesis. We speculate that delayed onset aspartate neurotoxicity might be related to apoptosis.
The histopathologic findings in group A (50 mmol/L aspartate with 10 minutes of isolation group) showed severely injured gray matter with neuronal and vessel necrosis. On the other hand, the white matter was spared even in the severely injured sections. These pathologic findings are typical in the setting of excitatory amino acid neurotoxicity reported by Redmond and colleagues [12]. Most of the neurons in group D (MK-801 treated before aspartate infusion group) were well preserved. These results supported the notion that the neuronal degeneration in this model is from aspartate excitotoxicity mediated through NMDA receptor activation, and MK-801 might act to block further production of glutamate that exerts excitotoxic effects.
Since Faden and Simon’s [13] report, accumulated evidence has shown that MK-801, a noncompetitive NMDA receptor antagonist, reduces neuronal injury in vivo. We carried out segmental infusion of 6 mg/kg MK-801 immediately after aortic clamping, because this model was designed for the use of NMDA receptor antagonists as a spinoplegia. However, lower doses of MK-801 were used for the preceding administration before the neuronal insult [12, 13]. We estimate that a higher dose is necessary to reach the protective level immediately after aortic clamping.
Olney and colleagues [14] reported short-term histopathologic change to neurons with vacuolation in rats treated with MK-801. NMDA receptor antagonists are reported to cause widespread central nervous system depression and learning impairment. We found minimal neuronal change with shrinkage in rabbits receiving MK-801; however, this finding was not identical to that reported as the neuronal injury induced by MK-801.
It was reported that selfotel, a competitive NMDA receptor antagonist, did not exhibit significant protective effect on the spinal cord against 60 minutes of ischemia in pig [15] because competitive NMDA receptor antagonists possess polar charges and they cannot readily penetrate the blood-brain barrier [16, 17]. In addition, the mechanism of ischemic spinal cord injury is multifactorial. Thus, it might be necessary to use the combined therapeutic methods to prevent ischemic neuronal injury.
We estimate that aspartate may enhance neurotoxic effect on neurons in prolonged ischemic or reperfusion phase that were damaged by glutamate in the early phase of ischemia. Moreover, we speculate that aspartate may be implicated in delayed onset paraplegia in the spinal cord. However, further studies are needed to evaluate the involvement of aspartate and glutamate in mediating neuronal injury in vivo. We conclude that our model may be useful in exploring the mechanism of the NMDA receptor mediated neurotoxicity and to evaluate the ability of protective agents to reduce neuronal injury in ischemic spinal cord.
|
Acknowledgments
|
|---|
We gratefully acknowledge the help of Mr Takashi Kimura with the pathologic analysis. We are also grateful to Ms Etsuko Doi for her technical assistance.
|
References
|
|---|
-
Mangano R.M., Schwarcz R. Chronic infusion of endogenous excitatory amino acids into rat striatum and hippocampus. Brain Res Bull 1983;10:47-51.[Medline]
-
Anderson K.J., Monaghan D.T., Bridges R.J., Tavoularis A.L., Cotman C.W. Autoradiographic characterization of putative excitatory amino acid transport sites. Neuroscience 1990;38:311-322.[Medline]
-
Mori A., Ueda T., Nakamichi T., et al. Detrimental effects of exogenous glutamate on spinal cord neurons during brief ischemia in vivo. Ann Thorac Surg 1997;63:1057-1062.[Abstract/Free Full Text]
-
Choi D.W., Viseskul V., Amirthanayagam M., Monyer H. Aspartate neurotoxicity on cultured cortical neurons. J Neurosci Res 1989;23:116-121.[Medline]
-
McDonald J.W., Johnston M.V. Physiological and pathophysiological roles of excitatory amino acids during central nervous system development. Brain Res Brain Rev 1990;15:41-70.[Medline]
-
Regan R.F. The vulnerability of spinal cord neurons to excitotoxic injury. Neuroscience Letters 1996;213:9-12.[Medline]
-
Simpson R.K., Robertson C.S., Goodman J.C. Spinal cord ischemia-induced elevation of amino acids. Neurochem Res 1990;15:635-639.[Medline]
-
Rokkas C.K., Helfrich L.R., Jr, Lobner D.C., Choi D.W., Kouchoukos N.T. Dextrorphan inhibits the release of excitatory amino acids during spinal cord ischemia. Ann Thorac Surg 1994;58:312-320.[Abstract]
-
Brock M.V., Redmond J.M., Ishiwa S., et al. Clinical markers in CSF for determining neurologic deficits after thoracoabdominal aortic aneurysm repairs. Ann Thorac Surg 1997;64:999-1003.[Abstract/Free Full Text]
-
Tseng E.E., Brock M.V., Lange M.S., et al. Nitric oxide mediates neurologic injury after hypothermic circulatory arrest. Ann Thorac Surg 1999;67:65-71.[Abstract/Free Full Text]
-
Tseng E.E., Brock M.V., Kwon C.C., et al. Increased intracerebral excitatory amino acids and nitric oxide after hypothermic circulatory arrest. Ann Thorac Surg 1999;67:371-376.[Abstract/Free Full Text]
-
Redmond J.M., Gillinov A.M., Zehr K.J., et al. Glutamate excitotoxicity. A mechanism of neurologic injury associated with hypothermic circulatory arrest. J Thorac Cardiovasc Surg 1994;107:776-787.[Abstract/Free Full Text]
-
Faden A.I., Simon R.P. A potential role for excitotoxins in the pathophysiology of spinal cord injury. Ann Neurol 1988;23:623-626.[Medline]
-
Olney J.W., Labruyere J., Price M.T. Pathological changes induced in cerebrocortical neurons by phencyclidine and related drugs. Science 1989;244:1360-1362.[Abstract/Free Full Text]
-
Sun J., Hirsch D., Svensson G. Spinal cord protection by papaverine and intrathecal cooling during aortic crossclamping. J Cardiovasc Surg 1998;39:839-842.[Medline]
-
Watkins J.C., Olverman H.J. Agonists and antagonists for excitatory amino acid receptors. Trends Neurosci 1987;10:265-272.
-
Kemp J.A., Foster A.C., Wong E.H.F. Non-competitive antagonists of excitatory amino acid receptors. Trend Neurosci 1987;10:294-298.
Accepted for publication April 26, 2000.
Top
Abstract
Introduction
