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Anesth Analg 2003;96:1777-1784
© 2003 International Anesthesia Research Society

NEUROSURGICAL ANESTHESIA

The Temporal Profile of the Reaction of Microglia, Astrocytes, and Macrophages in the Delayed Onset Paraplegia After Transient Spinal Cord Ischemia in Rabbits

Satoshi Matsumoto, MD, Mishiya Matsumoto, MD, Atsuo Yamashita, MD, Kazunobu Ohtake, MD, Kazuyoshi Ishida, MD, Yasuhiro Morimoto, MD, and Takefumi Sakabe, MD

Department of Anesthesiology-Resuscitology, Yamaguchi University School of Medicine, Japan

Address correspondence and reprint requests to Mishiya Matsumoto, MD, Department of Anesthesiology-Resuscitology, Yamaguchi University School of Medicine, 1–1-1 Minami-Kogushi, Ube, Yamaguchi 755–8505, Japan. Address e-mail to mishiya{at}yamaguchi-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
In the present study, we sought to elucidate the temporal profile of the reaction of microglia, astrocytes, and macrophages in the progression of delayed onset motor dysfunction after spinal cord ischemia (15 min) in rabbits. At 2, 4, 8, 12, 24, and 48 h after reperfusion (9 animals in each), hind limb motor function was assessed, and the lumbar spinal cord was histologically examined. Delayed motor dysfunction was observed in most animals at 48 h after ischemia, which could be predicted by a poor recovery of segmental spinal cord evoked potentials at 15 min of reperfusion. In the gray matter of the lumbar spinal cord, both microglia and astrocytes were activated early (2 h) after reperfusion. Microglia were diffusely activated and engulfed motor neurons irrespective of the recovery of segmental spinal cord evoked potentials. In contrast, early astrocytic activation was confined to the area where neurons started to show degeneration. Macrophages were first detected at 8 h after reperfusion and mainly surrounded the infarction area later. Although the precise roles of the activation of microglia, astrocytes, and macrophages are to be further determined, the results indicate that understanding functional changes of astrocytes may be important in the mechanism of delayed onset motor dysfunction including paraplegia.

IMPLICATIONS: Microglia and macrophages play a role in removing tissue debris after transient spinal cord ischemia. Disturbance of astrocytic defense mechanism, breakdown of the blood-spinal cord barrier, or both seemed to be involved in the development of delayed motor dysfunction.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Delayed onset paraplegia can develop in humans after transient aortic occlusion in thoracoabdominal aneurysm surgery (1). This type of paraplegia was also reported in rabbits 8–48 h after transient spinal cord ischemia (2–6). Jacobs et al. (7) demonstrated severe progressive breakdown of blood-spinal cord barrier integrity that developed many hours after transient spinal cord ischemia in rabbits. However, the mechanism of progressive breakdown of the barrier integrity is still unknown. Sakurai et al. (8) suggested that delayed and selective death of the motor neurons after transient spinal cord ischemia in rabbits was not necrotic but rather predominantly apoptotic. In contrast, our recent study demonstrated that delayed onset paraplegia was largely associated with necrotic (not apoptotic) cell death with prominent inflammatory cell infiltration (9).

Microglia, astrocytes, and macrophages are activated after central nervous system injuries (10). Microglia and macrophages not only contribute to repair of the tissue but also release toxic substances (11,12). Although both microglia and macrophages have a potential to deteriorate ischemic injury after transient spinal cord ischemia (13), the temporal profile of the reaction of these cells is not known. Astrocytes are one of the major components of the blood-brain (spinal cord) barrier and play major roles in ion homeostasis, excluding toxic substances, and releasing neurotrophic factors (14), whereas inducible nitric oxide synthase expressed in astrocytes may cause nitric oxide-mediated neuronal cell death (15).

Therefore, it is still debated whether such activation of microglia, astrocytes, and macrophages after transient ischemia is beneficial or detrimental (10). Also, little is known about the role of activation of microglia, astrocytes, and macrophages in the progression of delayed onset paraplegia. The present study sought to elucidate the temporal profile of the reaction of microglia, astrocytes, and macrophages in the progression of delayed onset paraplegia.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The protocol of this study was approved by the Ethics Committee for Animal Experiment at Yamaguchi University School of Medicine. Fifty-seven New Zealand white rabbits weighing 2.9 ± 0.4 (mean ± SD) kg were used in this study. The methods for surgical preparation, producing spinal cord ischemia, and postischemic management were almost the same as our previous studies (3–6).

Briefly, the rabbits were anesthetized in a plastic box with 5% sevoflurane in oxygen. An ear vein catheter was inserted for administration of fluid and drugs. After intubation of the trachea, the rabbit’s lungs were mechanically ventilated with 2%–3% isoflurane in 40% oxygen-60% nitrogen. Core temperature was monitored with a calibrated esophageal thermistor (Model MGA-III, Type 219; Nihon Koden, Tokyo, Japan). After skin infiltration with 0.25% bupivacaine, PE-60 catheters were inserted into both femoral arteries to measure blood pressure above and below the aortic occlusion. The right femoral catheter was advanced 3 cm into the abdominal aorta, whereas the other was advanced 17 cm. To estimate spinal cord temperature, the paravertebral muscle temperature at the level of L4-5 was monitored by a calibrated needle-type thermistor (Model PTC-201: Unique Medical, Tokyo, Japan). The paravertebral muscle temperature was controlled at {approx}38.0°C with a heating lamp and warming pad throughout the study.

In the right lateral decubitus position, the abdominal aorta at the level of left renal artery was retroperitoneally exposed. A PE-60 catheter was placed around the aorta immediately distal to the left renal artery. Then an occluder tube was tunneled to the skin for later occlusion of the aorta to produce spinal cord ischemia.

To monitor segmental spinal cord evoked potential (SSCEP), the left sciatic nerve was exposed and stimulated. SSCEPs were recorded in a bipolar fashion from the needle electrodes (L4-5 and L5-6 levels) using Neuropack Four Mini (Model MEB-5304; Nihon Koden) before ischemia and every 2 min during ischemia (for 15 min) and reperfusion (for 15 min). In the typical recording of SSCEP, the first two negative waves (N1 and N2) are presynaptic components, and the last two negative waves (N3 and N4) are postsynaptic ones (16). It is reported that the animals in which the ratio of the amplitude of N3 to N1 (N3:N1) returned to <70% of control at 120 min after reperfusion could be predicted to be paretic or paralyzed 48 h after reperfusion (16). In the current study, we calculated the ratio of N3:N1 at 15 min after reperfusion to predict the final neurologic outcome (48 h after reperfusion).

The rabbits were randomly assigned to 1 of the 7 groups depending on the time schedule of the final evaluation after ischemia or sham procedure: 2 h after ischemia (2-h group; n = 9), 4 h after ischemia (4-h group; n = 9), 8 h after ischemia (8-h group; n = 9), 12 h after ischemia (12-h group; n = 9), 24 h after ischemia (24-h group; n = 9), 48 h after ischemia (48-h group; n = 9), and sham operation (control group; n = 3).

After completion of surgery, end-tidal isoflurane concentration was maintained at 2%. Heparin 400 U was administered IV immediately before aortic occlusion. Spinal cord ischemia was produced for 15 min. In the control group, only surgical manipulation without aortic occlusion was performed. All catheters were then removed, and all incisions were sutured. Anesthetic administration was discontinued and extubation of the trachea was performed. The rabbits were allowed to recover in a warmed plastic box that contained supplemental oxygen for 6 h. IV fluid was provided until the rabbits began to drink. Antibiotic (cephazolin 30 mg/kg IM) was administered once daily.

The rabbits were neurologically assessed by an observer unaware of the treatment group using the 5-point score system proposed by Drummond and Moore (17): 4 = normal motor function, 3 = ability to draw legs under body and hop but not normally, 2 = some lower-extremity function with good antigravity strength but inability to draw legs under body, 1 = poor lower-extremity motor function but weak antigravity movement only, and 0 = paraplegic with no lower-extremity function.

After completion of neurologic function scoring at prescheduled times (2, 4, 8, 12, 24, or 48 h), the rabbits were reanesthetized with 2% isoflurane in oxygen. Transcardiac perfusion and fixation were performed with 10% phosphate-buffered formalin. Coronal sections of the spinal cord at the level of L5 were cut at a thickness of 8 µm. The sections were stained with hematoxylin and eosin, lectin histochemistry for microglia, or immunohistochemistry for glial fibrillary acidic protein (GFAP), and macrophages.

For lectin staining, the sections were deparaffinized using xylene and ethanol. Endogenous avidin-biotin binding was blocked (Bloking Kit, Vector, Japan). The sections were incubated for two days with biotinylated lectin (Biotin-RCA120, Honen, Japan) diluted to 2.5 µg/mL at 4°C. The sections were then reacted with an ABC kit (Vectastain ABC-Elite Kit; Vector) at room temperature for 60 min and visualized with 3,3'-diaminobenzidine hydrochloride. These sections were then counterstained with hematoxylin.

For immunohistochemistry, deparaffinized sections were used. Endogenous peroxidase was inactivated using 3% hydrogen peroxide in methanol. After rinsing in phosphate-buffered saline (pH value of 7.2; 0.1 mol/L), nonspecific protein binding was blocked with 10% normal goat serum. The sections were incubated for 18 h with monoclonal antibodies against GFAP (6F2; DAKO, Glostrup, Denmark) and macrophage (RAM11; DAKO) at 4°C. Antibodies dilutions in phosphate-buffered saline were 1:50 for 6F2 and 1:50 for RAM11. This was followed by incubation with the second antibody (Histofine simple stain PO(M); Nichirei, Chiba, Japan) at room temperature for 30 min and visualized with 3,3'-diaminobenzidine hydrochloride. These sections were then counterstained with hematoxylin.

We used two approaches for the quantification of our histological results: (a) counting activated microglia and macrophages in the gray matter of the anterior spinal cord (anterior to a line drawn through the central canal perpendicular to the vertical axis) and (b) rating the degree of astroglial reaction in the gray matter of the anterior spinal cord. Histological evaluation was performed with two sections for each animal by an observer unaware of the treatment groups. Activated microglia were identified by their enlarged size, stout processes, and intense staining. It was difficult to count the number of astrocytes. Therefore, the astroglial reaction was rated using the 5-point grading scale: 4 = strong, 3 = moderate, 2 = mild, 1 = partial/weak, and 0 = nil. A distinct GFAP staining without astrocyte hypertrophy was rated as moderate, and an intense GFAP staining with astrocyte hypertrophy was rated as strong. Physiologic variables were analyzed by a repeated-measures analysis of variance (ANOVA). P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
There were no significant differences among the groups for proximal mean arterial blood pressure, heart rate, esophageal temperature, paravertebral muscle temperature, PaO2, PaCO2, pH value, glucose, and hematocrit (Table). Distal mean arterial blood pressure values during ischemia were significantly decreased in the ischemia groups than those of the control group in which the aorta was not occluded. All rabbits survived until the prescheduled time for neurologic assessment and histological evaluations.


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  		Table 1. Physiologic Variables
 
Neurologic function score of hind limbs at the final time point in each group and the time course of function in the 48 h group are shown in Figure 1A and 1B, respectively. Hind limb motor function tended to show some improvement until 8 h after reperfusion, and thereafter, progressive deterioration was observed (Fig. 1A and 1B). Regarding the relationship between neurologic function and early SSCEP recovery in the 48-h group, the rabbits with N3:N1 recovery rate <70% all showed delayed motor dysfunction (Fig. 1B). All rabbits in the control group had normal motor function (score 4; not shown in Fig. 1).



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  		Figure 1. (A) Individual neurologic function scores after 15-min ischemia in 6 groups (2-, 4-, 8-, 12-, 24-, and 48-h group). Each symbol represents data for one rabbit. Neurologic function scores range from 0 (paraplegia) to 4 (normal). The segmental spinal cord evoked potential (SSCEP) recovery rate is the ratio of the amplitude of the third negative wave (N3) to the first negative wave (N1) (N3:N1) in percent at 15 min after reperfusion. An open circle represents the rabbit with N3:N1 recovery >=70%. A filled circle represents the rabbit with N3:N1 recovery <70%. (B) Changes of individual neurologic function scores after 15-min ischemia in the 48-h group. Each symbol represents data for one rabbit. Neurologic function scores range from 0 (paraplegia) to 4 (normal). An open circle represents the rabbit with N3:N1 recovery >=70%. A filled circle represents the rabbit with N3:N1 recovery <70%.

 
Microglia started to increase in number in all the areas of the gray matter at 2 h after reperfusion, regardless of the N1:N3 recovery rate (Fig. 2, A and B), with a gradual increase with time until 8 to 12 h after reperfusion (Fig. 2, C and D). Microglia were enlarged and showed stout processes and intense staining. They not only engulfed the motor neuron having degenerative changes (Fig. 2C), but also engulfed motor neurons having normal appearance (Fig. 2D). Microglial activation was more intense at 24 and 48 h after reperfusion in the rabbit with N3:N1 recovery <70% (Fig. 2, E and G), whereas the number of microglia and intensity of the activation were less in the rabbits with N3:N1 >=70% (Fig. 2, F and H). Figure 3 summarizes the number of microglia at each time point (Fig. 3A). In the rabbits with severe neurologic deficits (score 1 or 0) in the 24- and 48-h groups, most of the gray matter was destroyed (showing a large infarction). Microglia were not seen in the infarction core but seen in the surrounding area of the infarction. Microglia were only rarely and weakly stained with lectin in the gray matter of sham-operated rabbits (not shown).



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  		Figure 2. Microphotographs of the sections showing motor neurons and microglia in the spinal cord (L5) stained with lectin (original magnification x400). (A) The section from the rabbit with moderate motor dysfunction (score 2) and N3:N1 recovery <70%. The motor neurons still look normal. The activated microglia can be seen (2-h group). (B) The section from the rabbit with normal motor function (score 4) and N3:N1 recovery >=70%. The motor neurons look normal and the activated microglia can be seen (2-h group). (C) The section from the rabbit with moderate motor dysfunction (score 2) and N3:N1 recovery <70%. Finely granular dispersed Nissl substance can be seen in the motor neurons. The activated microglia engulf the degenerated motor neurons (8-h group). (D) The section from the rabbit with slight motor dysfunction (score 3) and N3:N1 recovery >=70%. The motor neurons look normal but are engulfed by activated microglia (8-h group). (E) The section from the animal with paraplegia (score 0) and N3:N1 recovery <70%. The normal structures of the ventral horn were destroyed and the number of activated microglia increased (24-h group). (F) The section from the animal with normal motor function (score 4) and N3/N1 recovery >=70%. The motor neurons look normal and the activation of microglia is not prominent (24-h group). (G) The section from the animal with moderate motor dysfunction (score 2) and N3:N1 recovery <70%. Microglia were seen in the surrounding the area of the infarction (not seen in the core) (48-h group). (H) The section from the animal with normal motor function (score 4) and N3:N1 recovery >=70%. The motor neurons look normal and the activation of microglia is not apparent (48-h group).

 


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  		Figure 3. The individual number of activated microglia (A) and macrophages (C) and the individual scale of glial fibrillary acidic protein (GFAP) intensity (B) in the L5 level of the anterior spinal cord (anterior to a line drawn through the central canal perpendicular to the vertical axis) in 6 groups (2-, 4-, 8-, 12-, 24-, and 48-h group). Each symbol represents data for one animal. An open circle represents the animal with N3:N1 recovery >=70%. A filled circle represents the animal with N3:N1 recovery <70%. (A) Microglia started to increase in number at 2 h after reperfusion with a gradual increase with time until 12 h after reperfusion, irrespective of the N1:N3 recovery rate. The number of activated microglia was larger at 24 and 48 h after reperfusion in the animal with N3:N1 recovery <70%, whereas the number of microglia was less in the animals with N3:N1 >=70%. (B) The intensity of GFAP was rated using the 5-point grading scale: 4 = strong, 3 = moderate, 2 = mild, 1 = partial/weak, 0 = nil. The intensity of GFAP started to increase at 2 h after reperfusion and dramatically increased at 24 to 48 h after reperfusion, especially in the animals with N3:N1 recovery <70%. (C) No or only a few macrophages were observed in the animals with N3:N1 recovery >=70% throughout the study period. Macrophages were first detected 8 h after reperfusion in the animals with N3:N1 recovery <70%. They increased in number at 24 to 48 h after reperfusion.

 
GFAP immunoreactivity in the 2-h group was mildly increased in all areas of the gray matter in 5 of 9 rabbits (Fig. 4, A and B), whereas the remaining 4 rabbits showed mild increase only in the intermediate area of the gray matter (Fig. 4C), not in the ventral horn (Fig. 4D). Further increase in GFAP immunoreactivity was not obvious until 12 h after reperfusion (Fig. 3B, 4E–H). GFAP immunoreactivity began to increase in the area where neurons started to show degeneration. At 24 h after reperfusion, GFAP staining became intense. Distinct hypertrophy of astrocytes was observed in 6 of 8 rabbits with N3:N1 <70% at 48 h after reperfusion. Astrocytic hypertrophy (scale 4) was observed in the infarction area (Fig. 4L). In contrast, GFAP immunoreactivity increased only mildly at 24–48 h after reperfusion in the rabbits with N3:N1 >=70% (Fig. 4J–N). Astrocytes were only weakly stained for GFAP in the gray matter in sham-operated animals (not shown).



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  		Figure 4. Microphotographs of the sections showing motor neurons and astrocytes in the spinal cord (L5) stained with glial fibrillary acidic protein (GFAP) immunohistochemistry (original magnification x400). (A and B) The section from the animal with moderate motor dysfunction (score 2) and N3:N1 recovery <70%. The intensity of GFAP started to increase mildly in both the intermediate area of the gray matter (A) and the ventral horn (B). Nissl substance of the motor neuron is already dispersed (B) (2-h group). (C and D) The section from the animal with normal motor function (score 4) and N3:N1 recovery >=70%. The intensity of GFAP increased in the intermediate area of the gray matter (C) but not obvious in the ventral horn (D) (2-h group). (E and F) The section from the animal with normal motor function (score 4) and N3:N1 recovery <70%. The intensity of GFAP mildly increased in both the intermediate area of the gray matter (E) and the ventral horn (F). Nissl substance of the motor neuron is already dispersed (F) (8-h group). (G and H) The section from the animal with slight motor dysfunction (score 3) and N3:N1 recovery >=70. The intensity of GFAP increased in the intermediate area of the gray matter (G) but not obvious in the ventral horn (H) (8-h group). (I) The section of the ventral horn from the animal with normal motor function (score 4) and N3:N1 recovery <70%. Although motor function looked normal, many motor neurons died, and the intensity of GFAP moderately increased. This animal would have developed delayed motor dysfunction at 48 h after reperfusion (24-h group). (J and K) The section from the animal with normal motor function (score 4) and N3:N1 recovery >=70%. The intensity of GFAP mildly increased in both the intermediate area of the gray matter (J) and the ventral horn (K) (24-h group). (L) The section of the ventral horn from the animal with paraplegia (score 0) and N3:N1 recovery <70%. Motor neurons disappeared, and the intensity of GFAP is strong. Hypertrophic astrocytes (arrows) can be seen (48-h group). (M and N) The section from the animal with normal motor function (score 4) and N3:N1 recovery >=70%. The intensity of GFAP mildly increased in both the intermediate area of the gray matter (M) and the ventral horn (N). The normal appearance of the motor neuron can be seen (48-h group).

 
Macrophages were not seen in the control rabbits (Fig. 5A). Macrophages were first detected 8 h after reperfusion in the rabbits with N3:N1 recovery <70% (Fig. 3C and 5B). They increased in number at 24–48 h after reperfusion (Fig. 3C) and mainly accumulated in the area surrounding the infarction (Fig. 5C) but were not seen in the infarction core. In the rabbits with N3:N1 recovery >=70%, no or only a few macrophages were observed throughout the study period (Fig. 3C and 5D).



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  		Figure 5. Microphotographs of the sections showing motor neurons and macrophages in the spinal cord (L5) stained with immunohistochemistry for macrophages (RAM 11) (original magnification x400). Macrophages (arrows) were identified by a brown-colored cell body. (A) Normal appearance of motor neuron can be seen. No macrophage is observed (Sham control [48 h after reperfusion]). (B) The section of the ventral horn from the animal with slight motor dysfunction (score 3) and N3:N1 recovery <70%. Degenerated motor neurons with dispersed Nissl substance can be seen. Macrophages were first detected at 8 h after reperfusion (8-h group). (C) The section of the ventral horn from the animal with severe motor dysfunction (score 1) and N3:N1 recovery <70%. Many macrophages accumulated in the surrounding area of the infarction (48-h group). (D) The section of the ventral horn from the animal with normal motor function (score 4) and N3:N1 recovery >=70%. Normal appearance of the motor neuron can be seen. Only a few macrophages are observed (48-h group).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The principal findings of the present study are as follows: (a) delayed motor dysfunction was observed at 48 hours after transient spinal cord ischemia in all rabbits with the N3:N1 recovery rate <70% (at 15 minutes after reperfusion), (b) microglial activation was seen early (two hours) after reperfusion in all areas of the gray matter, regardless of the predicted outcome, (c) an increase in GFAP immunoreactivity also began rapidly, starting in the area where neurons were thought to be in danger, and (d) infiltration of macrophages into the gray matter started to be observed later than eight hours after reperfusion.

The model used in the current study is the one that develops delayed motor dysfunction. We killed the rabbits at the prescheduled time points, namely at 2, 4, 8, 12, 24, and 48 hours after reperfusion. Therefore, whether the rabbits develop delayed motor dysfunction could be confirmed only in the 48-hour group. However, an earlier report demonstrated that the N3:N1 recovery rate <70% at 120 min after reperfusion could predict paretic or paralytic outcome by 48 h after reperfusion although this variable is principally reflecting sensory function (16). In the current study, we measured N3:N1 at 15 minutes after reperfusion to predict the final neurologic outcome (48 hours after reperfusion). If this variable satisfactorily predicts the final neurologic outcome, the relationship between earlier glial responses and possible development of delayed motor dysfunction can be evaluated in detail. Indeed, all rabbits with a N3:N1 recovery rate of <70% developed delayed motor dysfunction in the 48-hour group. Therefore, N3:N1 recovery <70% at 15 minutes after reperfusion seems to predict the delayed motor dysfunction with acceptable reliability.

Glial activation occurs in response to brain ischemia (10,18), and the response is very rapid, especially in microglia, which increase in number, for example, in the hippocampal CA1 sector as early as 20 minutes after reperfusion of forebrain ischemia (19). The precise pathophysiological role of microglial proliferation in ischemia is still largely unknown (20). Because an in vitro study showed that microglia released toxic substances and have cytotoxic properties (21), we hypothesized that activated microglia in the early reperfusion phase might be involved in the development of delayed motor dysfunction. However, in the present study, motor neurons engulfed by microglia were observed not only in the rabbits with N3:N1 recovery <70%, but also in the rabbits with N3:N1 recovery >=70%. Early microglial reaction seems to occur similarly in both animals that will later develop or will not develop delayed motor dysfunction. At 24–48 hours after reperfusion, gray matter structures were markedly destroyed, showing infarction, in the rabbits that developed severe motor dysfunction. Microglia surrounded the infarction area and were not seen in the core. We speculate that microglia actively remove tissue debris and die there, as suggested in the model of transient focal cerebral ischemia (22), and that they do not actively injure neurons.

Astrocytes can be identified immunohistochemically by staining for the intermediate filament (GFAP) (23). GFAP is constitutively expressed in fibrous astrocytes of the white matter but at much smaller levels in protoplasmic astrocytes, which account for most of the astrocytes in the gray matter (10). Although inducible nitric oxide synthase expressed in astrocytes might cause nitric oxide-mediated neuronal death (15), activated astrocytes may increase the uptake of glutamate, provide lactate as an energy source (24), and release neurotrophic factors including nerve growth factor (25). It has been reported that GFAP has an important role in the survival of neurons after cerebral ischemia because the animals lacking GFAP have been shown to exhibit increased infarction after ischemia than the wild-type animals (26). In the present study, compared with the widespread activation of microglia in the early reperfusion period, the increase in GFAP immunoreactivity in the gray matter (protoplasmic astrocytes) was more selective and prominent in the area where neurons were thought to be in danger. We speculate that ischemia initiates the functional activation of astrocytes, which leads to an increased GFAP immunoreactivity and plays a role in protecting neurons and that neuronal death occurs when the severity of cellular derangement exceeds the protective action of astrocytes.

The appearance of macrophages was delayed compared with the response of microglia and astrocytes and was first detected eight hours after reperfusion. Macrophages increased in number in the rabbits with delayed motor dysfunction. Jacobs et al. (7) reported the progressive breakdown of the blood-spinal cord barrier at 8–24 hours after 25-minute ischemia. Therefore, it is likely that the breakdown of the blood-spinal cord barrier enables macrophages to migrate into the spinal cord. Macrophages mainly surrounded the infarction area at 24–48 hours after reperfusion, and they did not migrate into the ventral horn, even in the rabbits with N3:N1 recovery <70%, until apparent neuronal death occurred. Although pharmacological inhibition of mononuclear phagocytes was reported to reduce ischemic injury in the spinal cord (13), our observation suggests that macrophages do not cause motor neuron injury but eliminates tissue debris.

In summary, microglia and astrocytes responded quickly after transient spinal cord ischemia, whereas macrophages showed delayed response. Microglia and macrophages seem to play a role in eliminating tissue debris. Early astrocytic activation was more selective compared with the widespread activation of microglia and prominent in the area where neurons were thought to be in danger, possibly involved in protecting neurons there. Although further studies are required, the functional changes of astrocytes may be an important clue for elucidating the mechanism of delayed onset motor dysfunction including paraplegia.


   Acknowledgments
 
Supported, in part, by the Ministry of Education, Science, Sports, and Culture grant no. 11470323 (to T. Sakabe).

The authors thank Dr. Toshikazu Gondo (First Department of Pathology) for his advice in the assessment of histopathology.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication February 12, 2003.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press