Paraplegia is a devastating complication of thoracoabdominal aortic operations, with the reported incidence ranging from 2.4% to 40% (1–4). The mechanisms of postoperative paraplegia involve both acute ischemic and delayed reperfusion injury. Several attempts using hypothermia, cerebrospinal fluid drainage, N-methyl-d-aspartate receptor antagonists, calcium channel blockers, and free radical scavengers have been made to protect the spinal cord against ischemic injury (2,5,6). However, because their efficacy is limited, further investigation into the mechanisms of spinal cord injury are necessary to allow identification of more effective therapeutic interventions.
For the assessment of spinal cord injury after spinal cord ischemia (SCI), gray matter injury has been mainly focused upon as a target for therapy because of the traditional view that white matter is less vulnerable to ischemic injury. In fact, most published studies regarding SCI have assessed only neurologic and histologic severity of gray matter injury (7–9). However, evidence indicates the importance of white matter injury after SCI. (10–12). Follis et al. (10) demonstrated that white matter was more vulnerable to ischemia compared with gray matter in a rat model of SCI. Kanellopoulos et al. (12) indicated that assessment of gray matter injury only may not be sufficient as an indicator of spinal cord injury and, therefore, both gray and white matter should be assessed.
Although there have been a number of investigations regarding gray matter injury after SCI, data on the white matter injury are limited. Specifically, the relationship between the magnitude of gray and white matter injury has not been extensively examined. In the present study, we quantitatively assessed gray and white matter injury after SCI in rats, and the relationship between the magnitude of gray and white matter injury was determined. White matter injury was assessed based on the extent of vacuolation in hematoxylin and eosin (HE)-stained sections and accumulation of amyloid precursor protein (APP) by immunohistochemistry.
The study was approved by the Animal Experiment Committee of Nara Medical University (Kashihara, Nara, Japan). Twenty-five male Sprague-Dawley rats (Japan SLC, Inc., Shizuoka, Japan) weighing 350–450 g were used in the study. The rats were housed and maintained on a 12-h light-dark cycle with free access to food and water. All animals were neurologically intact before anesthesia and surgery.
SCI was induced as reported by Taira and Marsala (13). The rats were anesthetized in an acrylic box with isoflurane in an air/oxygen mixture. After induction, the trachea was intubated and the lungs were ventilated mechanically with 1.5% isoflurane in an air/oxygen mixture. Rectal temperature was continuously monitored (Mon-a-Therm; Mallinckrodt, St. Louis, MO) and maintained at 37 ± 0.5°C with an underbody heating pad and heat lamps. For monitoring distal arterial blood pressure and collecting blood specimens, a polyethylene catheter (PE-50) was inserted into the tail artery. A 2F Fogarty balloon catheter was inserted via the left femoral artery to the descending thoracic aorta so that the tip of the catheter reached the level of the left subclavian artery (10.5–11 cm from the site of insertion). A PE-60 catheter was inserted into the left carotid artery and connected to an external blood reservoir positioned at 54-cm height measured from the level of the rat’s body in order to reduce mean arterial blood pressure (MAP) proximal to the occlusion site during aortic occlusion to the target of 40 mm Hg. After all cannulas were placed, 200 U of heparin was injected into the tail artery.
After surgical preparation, the animals were randomly allocated to one of the following 3 groups: animals who received SCI for 12 min (SCI-12; n = 8) or 15 min (SCI-15; n = 9), or those who underwent sham operation (n = 8). To induce SCI, the balloon catheter was inflated with 0.05–0.1 mL of saline, and blood was allowed to flow to the external reservoir. The completeness of occlusion was documented by an immediate and sustained loss of any detectable pulsation and a decrease in pressure below the level of aortic occlusion. After 12 or 15 min of ischemia, the balloon was deflated, and blood was reinfused over 30 s. Then the catheters were removed and incisions were closed. Protamine sulfate (4 mg) was administered subcutaneously before animals recovered from anesthesia. Blood gases, pH, and hematocrit were measured 10 min before and 10 min after the ischemia with a blood gas analyzer (GEM Premier; Mallinckrodt, Ann Arbor, MI). In the sham group, all catheters were inserted in the same manner, but the balloon was not inflated.
Neurologic Quantification of Ischemic Damage
An observer who was blinded to the experimental procedures carefully examined hindlimb motor function of the rats 24 h after reperfusion. Hindlimb motor function was assessed using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale (14). The scale has 22 values, which range from 0 (total paralysis) to 21 (normal locomotion). Values in the range of 1–8 are scored for small or large movements of the 3 hindlimb joints without plantar weight support or dorsal stepping, a score of 9 involves attainment of plantar weight support or dorsal stepping, and scores of 10–20 are given for progressive improvements in coordinated walking ability. The scores for both hindlimbs were averaged to obtain the score for each measurement.
Histologic Quantification of Ischemic Damage
Twenty-four hours after reperfusion, rats were anesthetized with isoflurane, and 3 mg/kg thiopental was administered intraperitoneally. The rats were then transcardially perfused with 100 mL of heparinized saline, followed by 150 mL of 3.7% formaldehyde. Twenty-four hours after perfusion, the spinal cords were removed and postfixed in the same fixative for 1–2 days. After this period, the fourth lumbar spinal segment was dissected, embedded in paraffin, and sectioned at 3 μm. These sections were stained with HE for quantitative evaluation.
Gray matter damage was assessed on the basis of the number of normal neurons per microscopic field in the ventral horn (laminae 7, 8, and 9) with ×200 magnification (Fig. 1). Cells that contained Nissl substance in the cytoplasm, loose chromatin, and prominent nucleoli were considered to be normal neurons. White matter damage was assessed on the basis of the extent of vacuolation in the ventral and ventrolateral white matter. The percentage of areas of vacuolation of total target area (0.04 mm2) was calculated (Fig. 1). Total area was divided into 144 subareas for ventral and ventrolateral white matter. Then the number of subareas, in which vacuoles occupied >75% of the subarea, was counted and the percentage of the number of subareas with vacuoles of total number of subareas was calculated as percentage area of vacuolation. The number of normal neurons and percentage area of vacuolation were taken as the average of the right and left hemicords in three consecutive sets of specimens from each animal.
Adjacent sections were immunostained with APP (Clone 22C11; Chemicon, Temecula, CA) to label ischemically damaged axons using the streptavidin-biotin method as instructed in the kit (Histofine SAB-PO [M] Kit; Nichirei Corp., Tokyo, Japan). Briefly, sections were deparaffinized and pretreated for antigen retrieval by a microwave oven in a citric acid buffer (10 mmol/L; pH 6.0) for 10 min for immunostaining, allowed to cool to room temperature, and rinsed in phosphate-buffered saline (PBS). To block endogenous peroxidase, sections were incubated in 3% hydrogen peroxide in methanol for 30 min, rinsed in PBS, and incubated again with normal rabbit serum for 10 min. The sections were rinsed and incubated at 4°C overnight with the primary mouse monoclonal antibody against APP diluted 1:500 in normal rabbit serum. The next day, the sections were rinsed, and exposed to a biotin-labeled rabbit antimouse immunoglobulin (Ig)G/IgA/IgM mixture for 5 min at room temperature, followed by streptavidin for 5 min. Copious washing with PBS between each step was essential. Antigen-antibody complex was visualized via 3,3′-diaminobenzidine (Nichirei Corp.) for 3 min. At the end of the procedure, the sections were lightly counterstained with hematoxylin, dehydrated through graded alcohols, and cleared in xylene before being coverslipped for microscopic examination. Negative controls for APP antibodies were performed by omitting the primary antibody. APP immunoreactivity was assessed in the areas of ventral and ventrolateral white matter as mentioned above. To assess the APP immunoreactivity, a score of 0 (no APP accumulation) or 1 (APP accumulation in axonal swellings) was assigned to each area and the total score in each animal was calculated (from 0 to 2). Investigators without knowledge of the injury models (sham or ischemia) performed the histologic assessment of the gray and white matter injury.
Differences in physiologic values, the number of normal neurons, and percentage area of vacuolation among the groups were assessed using one-way analysis of variance, and when significant differences were identified, the Student-Newman-Keuls test was performed for intergroup comparisons. The comparisons of percentage area of vacuolation within the group were performed using paired t-test. BBB and APP scores were analyzed using a nonparametric method (Kruskal-Wallis test) followed by the Mann-Whitney U-test. To determine the relationship between the number of normal neurons and percentage area of vacuolation, linear regression analysis was performed. To determine the relationship between BBB score and the number of normal neurons or percentage area of vacuolation, Spearman’s correlation analysis was performed. A P value < 0.05 was considered statistically significant. Physiologic variables, the number of normal neurons, and percentage area of vacuolation were expressed as means ± sd, and BBB and APP scores were expressed as medians with interquartile ranges in parentheses.
Demographic variables are shown in Table 1. There were no significant differences in pH, arterial carbon dioxide partial pressure (Paco2), arterial oxygen partial pressure (Pao2), hematocrit, glucose, distal MAP, and rectal temperature before ischemia among the groups. During ischemia, distal MAP values in the SCI-12 and SCI-15 groups were significantly lower compared with those in the sham group (P < 0.05). Rectal temperature during ischemia was similar among the groups. After reperfusion, pH values in the SCI-12 and SCI-15 groups were significantly lower compared with those in the sham group (P < 0.05). Paco2, Pao2, hematocrit, glucose, distal MAP, and rectal temperature after the reperfusion were similar among the groups.
Median values (25–75th) of BBB scores in the sham, SCI-12, and SCI-15 groups were 21 (20.5–21), 14.3 (5–16.5), and 2.0 (0–11.1), respectively. BBB scores in the SCI-12 and SCI-15 groups were significantly lower compared with those in the sham group (P < 0.05). The number of normal neurons in the SCI-12 and SCI-15 groups were significantly lower compared with those in the sham group (P < 0.05) (Fig. 2a). The number of normal neurons in the SCI-15 group was significantly less than that in the SCI-12 group (P < 0.05). The relationship between BBB scores and the number of normal neurons is shown in Figure 2b. There was a significant positive correlation between BBB scores and the number of normal neurons (with sham group: Spearman’s correlation coefficient 0.85, P < 0.0001; without sham group: Spearman’s correlation coefficient 0.85, P = 0.0006).
Figure 3 shows the results of white matter injury in the ventral and ventrolateral areas. Percentage areas of vacuolation in the SCI-12 and SCI-15 groups were significantly larger compared with those in the sham group (P < 0.05). Percentage areas of vacuolation in the SCI-15 group were significantly larger than those in the SCI-12 group (P < 0.05). In the SCI-15 group, percentage areas of vacuolation in the ventrolateral area were significantly larger compared with those in the ventral areas (P < 0.05). Representative photomicrographs of HE-stained sections in the ventrolateral white matter are shown in Figure 4. Whereas vacuolation was not noted in the sham group, vacuolation was widespread and prominent in the ventrolateral white matter in the SCI-15 group. The relationship between BBB scores and average percentage areas of vacuolation is shown in Figure 5a. There was a significant negative correlation between BBB scores and average percentage areas of vacuolation (with sham group: Spearman’s correlation coefficient −0.85, P < 0.0001; without sham group: Spearman’s correlation coefficient −0.56, P = 0.0316). Figure 5b shows the relationship between the number of normal neurons and average percentage areas of vacuolation. A significant negative correlation was noted between the number of normal neurons and average percentage areas of vacuolation (with sham group: r = 0.83, P < 0.0001; without sham group: r = 0.726, P = 0.01).
Immunohistochemical analysis revealed that APP immunoreactivity was accumulated in the regions of axon bundles in the ventral and ventrolateral white matter predominantly in the SCI-15 group, whereas accumulation of APP immunoreactivity was not noted in the sham group. Figure 4 shows the representative photomicrographs of APP immunohistochemistry in the ventrolateral areas of white matter. Median values of APP scores (25–75th) in the sham, SCI-12, and SCI-15 groups were 0 (0–0), 0.5 (0–1), and 2 (1.8–2.0), respectively. APP scores in the SCI-15 group were significantly higher compared with those in the SCI-12 and sham groups (P < 0.05).
The results in the present study show that SCI induced vacuolations in the ventral and ventrolateral areas of white matter as well as a decrease in the number of normal neurons. Immunohistochemical analysis revealed increased APP immunoreactivity in swollen axons in the white matter. These data indicate that both gray matter and white matter may be injured after SCI in rats. The magnitudes of white matter injury in the ventral and ventrolateral areas were correlated with the severity of gray matter injury.
In the present study, we used the rat SCI model, originally reported by Taira and Marsala (13). These researchers demonstrated that by decreasing the proximal aortic pressure to 40 mm Hg during aortic occlusion in rats, 10–12 minutes of aortic occlusion induced acute paraplegia. With 12 minutes of SCI, all animals displayed acute and persistent paraplegia, which remained unchanged for 2 days, and extensive necrotic changes and irregular cavitation of gray matter were mainly observed between laminae III to VII in the L2 to L5 segments. Those findings are consistent with the results in the present study, in which 12 and 15 minutes of ischemia induced acute paraplegia or paraparesis and a decrease in the number of normal neurons in the gray matter. However, in the study by Taira and Marsala (13), the magnitude of white matter injury was not evaluated.
Kanellopoulos et al. (12) evaluated both gray and white matter injury after 11 minutes of SCI in rats. In the gray matter, many neurons showed features characteristic of ischemic cell death, including cytoplasmic eosinophilia with disintegration of cytoarchitecture and nuclear pyknosis at 2 days after reperfusion. In the white matter, vacuolation was widespread and was prominent in the ventral and ventrolateral white matter at 2 days after ischemia. At 6 weeks after the insult, a loss of 47% and 58% of axons was observed in the ventral and ventrolateral white matter areas, respectively. Follis et al. (10) investigated the effects of 10–12 minutes of SCI on gray and white matter injury in rats and demonstrated that paraplegic animals had myelin vacuolation and an increased number of macrophages in the anterior and lateral columns as white matter injury with or without neuronal loss with gliosis in the gray matter. In a model of focal cerebral ischemia, Pantoni et al. (15) evaluated white matter injury in rats using light and electron microscopy and demonstrated that vacuolation and pallor of the white matter were substantial at 24 hours after ischemia and reflected the segmental swelling of myelinated axons, the formation of spaces between myelin sheaths and axolemma, and astrocyte swelling. These findings are compatible with the results in the present study, in which vacuolations in the ventral and ventrolateral areas of white matter were observed 24 hours after ischemia.
Because APP is transported by fast anterograde axonal transport, the accumulation of APP at sites of injury, accompanied by morphological evidence of axonal damage in the form of axonal swelling or bulbs, has been regarded as evidence of axonal injury. For the detection of axonal injury, APP accumulation has been used in models of cerebral ischemia (16–21) and spinal cord trauma (22). Several investigators have reported that damaged axons had a bulbous or swollen appearance with increased APP immunoreactivity within subcortical white matter and myelinated fiber tracts 24 hours after focal cerebral ischemia in rats (20,23). Westergren et al. (22) indicated that APP accumulation was noted in the swollen axons in the white matter 24 hours after spinal cord compression injury in rats. There have been few reports using APP immunoreactivity for the assessment of white matter injury after SCI. In the present study, we also noted the increased APP immunoreactivity in the swollen axons especially in animals with 15 minutes of SCI, which may support the findings of vacuolations obtained by HE staining.
Several investigators have reported the correlation between the degree of gray matter injury and hindlimb motor function after SCI (13,24). In the present study, we also observed the correlation between the number of normal neurons and BBB score. However, there has been little information regarding the relationship between the degree of white and gray matter injury and between the degree of white matter injury and hindlimb motor function after SCI. Follis et al. (10) evaluated 21 rats subjected to SCI and compared gray and white matter injury to neurologic function. They suggested that white matter was more vulnerable to SCI compared with gray matter. However, in their study, the criteria for white and gray matter injury were not clear. In contrast, in the present study, for the quantitative analysis of gray and white matter injury, the number of normal neurons and percentage areas of vacuolations were used respectively. The degree of white mater injury was correlated with the severity of gray matter injury and hindlimb motor function. However, the correlation between the BBB score and the number of normal neurons was better than the correlation between the BBB score and percentage areas of vacuolations, suggesting that gray matter injury may be a better indicator of hindlimb motor function compared with white matter injury.
There are several limitations in the present study that merit comment. First, assessments were performed only 24 hours after reperfusion. To determine the permanent effects of SCI on white matter injury, long-term assessments are required (25,26). Second, 12- and 15-minute intervals of SCI were selected. Because these constitute moderate-to-severe ischemia insult, the effects of mild ischemia were not systematically examined. Third, the corticospinal tract might be better for definition of the correlation between white matter injury and hindlimb motor function. However, electron microscopy is required for proper assessment of corticospinal tract injury in rats, whereas assessments of ventral and ventrolateral white matter can be performed using light microscopy (11). We therefore only assessed ventral and ventrolateral white matter. Finally, white matter injury after SCI may be limited to the rat model. There have been few reports regarding white matter injury in other species.
In summary, we investigated gray and white matter injury in a rat model of SCI. SCI induced vacuolations with increased APP immunoreactivity in the ventral and ventrolateral areas of white matter as well as a decrease in the number of normal neurons, indicating that SCI induced both white and gray matter injury. The results also indicated that the degree of white matter injury was correlated with the severity of gray matter injury after a relatively short recovery period. Because the mechanisms of degeneration of white matter have been shown to differ from those of gray matter, strategies to reduce white matter injury may be different from those for gray matter. In this regard, assessment of both white and gray matter would be crucial in future experiments to evaluate therapeutic efficacy against ischemic spinal cord injury.
The authors thank Drs. Piyush M. Patel, John C. Drummond, Osamu Kakinohana, and Joho Tokumine, Department of Anesthesiology, University of California, San Diego, CA, and Dr. Akio Wanaka, Second Department of Anatomy, Nara Medical University, Nara, Japan, for their support.
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