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The Neuroprotective and Anti-inflammatory Effects of Diltiazem in Spinal Cord Ischaemia −Reperfusion Injury I Fansa, ME Altug, I Melek, E Ucar, T Kontas, B Akcora, E Atik and T Duman Journal of International Medical Research 2009 37: 520 DOI: 10.1177/147323000903700228 The online version of this article can be found at: http://imr.sagepub.com/content/37/2/520

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The Journal of International Medical Research 2009; 37: 520 – 533

The Neuroprotective and Anti-inflammatory Effects of Diltiazem in Spinal Cord Ischaemia–Reperfusion Injury I FANSA1, ME ALTUG6, I MELEK2, E UCAR3, T KONTAS7, B AKCORA4, E ATIK5 AND T DUMAN2 1

Department of Cardiovascular Surgery, 2Department of Neurology, 3Department of Internal Medicine, 4Department of Paediatric Surgery, and 5Department of Pathology, Faculty of Medicine, Mustafa Kemal University, Antakya/Hatay, Turkey; 6Department of Surgery, and 7Department of Biochemistry, Faculty of Veterinary Medicine, Mustafa Kemal University, Antakya/Hatay, Turkey

The protective effects of diltiazem were examined in a rabbit model of spinal cord ischaemia–reperfusion induced by infrarenal aortic occlusion for 30 min. In the diltiazem group (n = 6), an intravenous infusion (2 µg/kg per min) was started 10 min before ischaemia induction; normal saline solution was infused in the control group (n = 6). Neurological function was assessed using modified Tarlov criteria 24 h after surgery. Plasma samples were analysed for interleukin (IL)6 and IL-10. Spinal tissue was analysed for malondialdehyde, nitric oxide and reduced glutathione activities. Tarlov KEY WORDS: DILTIAZEM; SPINAL

scores of the diltiazem-treated rabbits indicated significantly improved hindlimb motor function compared with the control group. The diltiazem group also had better quantitative and qualitative histopathological findings. Diltiazem infusion significantly reduced IL-6 levels 3 and 24 h after reperfusion compared with the control group. The mean IL-10 level in the diltiazem group was significantly higher than in the control group 24 h after reperfusion. It is concluded that diltiazem has cytoprotective and anti-inflammatory properties, leading to reduced spinal cord injury.

CORD ISCHAEMIA;

FREE

ANIMAL

MODEL; INFLAMMATORY CYTOKINES;

RADICALS

Introduction The spinal cord is sensitive to ischaemia, and paraplegia that results from spinal cord ischaemia is a catastrophic complication of thoraco-abdominal aortic surgery. Spinal cord injury results from both ischaemia and

the ensuing reperfusion. Neurological injury due to ischaemia–reperfusion (I/R) injury of the spinal cord has an incidence of between 2.9% and 23%.1 Despite several surgical modifications and pharmacological approaches, post-operative spinal cord

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I Fansa, ME Altug, I Melek et al. Neuroprotective and anti-inflammatory effects of diltiazem dysfunction has not been totally eliminated.2 Although the exact pathophysiological mechanisms that underlie spinal cord I/R injury remain to be defined, varying membrane permeability to calcium influx into the ischaemic neuron secondary to excitotoxic stimulus is known to play an important role. At the initial stage of the ischaemic cascade, sodium influx causes cellular depolarization that contributes to the activation of calcium-dependent glutamate release which, in turn, leads to excessive calcium entry through voltagegated calcium channels. Intracellular hypocalcaemia leads to the activation of many proteases and the production of reactive oxygen species, and contributes to neuronal damage.3 – 5 Calcium accumulation in the spinal cord was shown during and after ischaemia lasting > 20 min, which correlated with irreversible spinal cord injury. 6 The blockade of this calcium influx could, therefore, lower the metabolic burden, thus decreasing I/R injury. Indeed, some studies have attempted to examine the effects of calcium channel antagonists in spinal cord I/R injury. 7 – 12 Flunarizine improved neurological recovery when administered before ischaemia in experimental spinal cord ischaemia in rabbits.9,10 Nimodipine has been evaluated in spinal cord ischaemia settings, with controversial results. For example, nimodipine administered during and after ischaemia in a canine spinal cord model gave no significant benefit with regard to histological damage and neurological recovery.7 Conversely, nimodipine protected the spinal cord during ischaemic events when administered in sufficient doses in pigs. 11 In a rat model of spinal cord ischaemia, animals treated with intrathecal ziconotide had better motor function and

fewer histopathological changes compared with controls.12 Finally, reduced hind-limb motor dysfunction was demonstrated in rabbits treated with the diphenylpiperazine calcium channel antagonists, MK-801 and KB-2796.8 To our knowledge, no published studies have examined the effects of diltiazem on spinal cord I/R injury in an animal model. Diltiazem is a calcium channel antagonist in wide clinical use. Besides its vasodilatory effects on blood vessels, various biological and metabolic actions of diltiazem have been well documented. For example, it has several features related to protection of the myocardium and liver against I/R injury in animal models.13 – 19 Ultrastructural studies of rat hearts injured by I/R indicated that diltiazem prevents intracellular calcium overload, protects mitochondrial integrity and the lipid fraction within the cell membrane against the toxic effects of free oxygen radicals, and preserves high-energy phosphates and membrane permeability.13 – 17 Additionally, diltiazem has been shown to have a protective effect by maintaining low calcium levels in hepatocytes in liver I/R injury19 and it also has anti-inflammatory actions.20,21 These findings led us to the hypothesis that diltiazem, a calcium channel blocking agent, would provide neurological protection during I/R of the spinal cord. The present study investigated the therapeutic effect of diltiazem on I/R injury of the rabbit spinal cord. To assess the ability of diltiazem to prevent neurological injury, hind-limb motor function was evaluated and the spinal cord was examined by quantitative and qualitative histopathology. Furthermore, the effects of diltiazem administration on inflammation were evaluated by measuring interleukin (IL)-6 and IL-10 plasma concentrations.

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I Fansa, ME Altug, I Melek et al. Neuroprotective and anti-inflammatory effects of diltiazem

Materials and methods ANIMALS Male New Zealand White rabbits were used in this study. The experimental protocols were approved by the Animal Research Committee of the Faculty of Veterinary Medicine of Mustafa Kemal University. The animals were housed in individual cages in a temperature and light–dark controlled environment with free access to food and water. All animals received humane care in accordance with the Guide for the Care and Use of Laboratory Animals.22

EXPERIMENTAL PROCEDURES Anaesthesia was induced by intramuscular injection of 5 mg/kg xylazine hydrochloride (Rompun®; Bayer, Istanbul, Turkey) and 40 – 50 mg/kg ketamine hydrochloride (Ketalar®; Pfizer, Istanbul, Turkey). General anaesthesia was maintained by inhalation of 1 – 3% isoflurane (Aerrane®; Baxter, Unterschleissheim, Germany) in oxygen with a flow rate of 2 l/min via a home-made facemask device. The ear vein and artery were cannulated before surgery. Arterial pressure and heart rate were monitored continuously. Body temperature was measured by rectal thermometry and maintained at 37 °C with a pad and heating lamp. The animals were randomly divided into three groups and placed in the supine position. A midline laparotomy was performed after the skin had been shaved and the surgical site had been prepared with 10% povidone–iodine solution. The intestines were deflected to the right and the infrarenal abdominal aorta was isolated from the left renal artery down to the aortic bifurcation. In all groups except the sham group (n = 4) in which the operation was performed without aortic occlusion, spinal cord

ischaemia was induced by infra-renal aortic occlusion for 30 min. The aorta was crossclamped at two sites (one just below the left renal artery and the other just above the aortic bifurcation) and the aorta was palpated to confirm loss of pulse. In the animals randomized to diltiazem (n = 6), an intravenous infusion of diltiazem (2 µg/kg per min) was started 10 min before aortic occlusion and continued for 60 min. In the animals randomized to the control group (n = 6), normal saline solution was infused during the same period. Each animal in the control and diltiazem groups was also given a dose of heparin (400 IU/kg) intravenously before aortic clamping. After removal of the aortic clamps, the abdomen was closed in layers and the animals were allowed to recover in their cages with free access to food and water.

BIOCHEMICAL ANALYSES Blood samples were centrifuged at 3000 rpm (NF 200 centrifuge; Nuve, Ankara, Turkey) for 10 min to obtain plasma. All samples were stored at −20 °C until use. Plasma samples were analysed for IL-6 and IL-10. Malondialdehyde (MDA), nitric oxide (NO) and reduced glutathione (GSH) were measured in spinal tissue samples. For quantitative measurement of IL-6 and IL-10 in plasma samples, enzyme-linked immunosorbent assay (ELISA) kits were used (Cat. Nos. KHCOO61 and KHC0101, respectively; Invitrogen Biosource™, Carlsbad, CA, USA).23,24 The kits are a solidphase sandwich ELISA in which the wells of microtitre strips are coated with a monoclonal antibody. To estimate spinal tissue antioxidant levels, tissue samples were prepared at 4 °C. Tissues were weighed and cut into small pieces. A 10% homogenate was prepared in 50 mM ice-cold phosphate-buffered saline

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I Fansa, ME Altug, I Melek et al. Neuroprotective and anti-inflammatory effects of diltiazem (PBS, pH 7.4) containing 5 mM ethylenediamine tetra-acetate with an ultrasonic homogenizer (UV 2070; Bandelin Electronics, Berlin, Germany). The homogenates were centrifuged at 15 000 rpm (NF 1200R centrifuge; Nuve) for 10 min at 4 °C and supernatants were obtained to estimate MDA, NO and GSH 24 h after I/R. Total protein levels in homogenates were estimated by the biuret method to determine the level of each parameter per gram of tissue protein.25

EVALUATION OF NEUROLOGICAL DEFICIT Neurological deficit scores were evaluated 24 h after surgery. Motor function of the hind limbs was scored from 0 to 5 according to a modified Tarlov scale 26 by an investigator blinded to the experimental group, as follows: 0, spastic paraplegia with no movement of the hind limbs; 1, spastic paraplegia with slight movement of the hind limbs; 2, good movement of the hind limbs but unable to stand; 3, able to stand, but unable to hop; 4, able to hop but wobbly and might fall on their side occasionally; 5, complete recovery, able to hop normally.

HISTOPATHOLOGICAL EVALUATIONS All rabbits were killed with using sodium pentobarbital (100 mg/kg body weight, IV) after neurological evaluation. The lumbar spinal segment was immediately obtained and flash-fixed in 10% buffered formalin. Segments were embedded in paraffin and serial transverse sections were cut (4 µm) for haematoxylin and eosin staining. Histopathological evaluations were performed by a pathologist blinded to the study groups using a light microscope (BX51; Olympus, Tokyo, Japan) at ×200

magnification. Features consistent with neuronal injury were to include eosinophilic cytoplasm, vacuolization and pycnotic appearance (loss of nuclear structure). Cells that contained Nissl substance in the cytoplasm, loose chromatin and prominent nucleoli were considered viable. A quantitative index of neuronal viability was calculated as the number of clearly viable neurons divided by the total neuronal count within the entire microscopic section for each animal. 27 Histological damage was scored qualitatively using a system developed by Caparrelli et al.28 A score of 0 – 4 was assigned to each section as follows: 0, frank necrosis; 1 severe cellular damage; 2 moderate cellular damage; 3 mild cellular damage; 4 normal histological appearance.

STATISTICAL ANALYSES Statistical analyses were performed with the SPSS® program version 13.0 (SPSS Inc., Chicago, IL, USA). All data were expressed as mean ± SD. The inflammatory variables and values for time to event were compared between groups using one-way analysis of variance (ANOVA) for repeated measures. If statistically significant effects were found, Tukey’s post hoc test was used. Changes within each group were compared using oneway ANOVA for repeated measures, followed by the Bonferroni test when appropriate. The histological evaluations and neurological deficit scores were analysed by Kruskal–Wallis ANOVA on ranks followed by the Tukey test. A P value < 0.05 was considered statistically significant.

Results PHYSIOLOGICAL PARAMETERS A total of 16 male New Zealand White rabbits, weighing 2.14 ± 0.38 kg were used. All animals survived the observation period.

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I Fansa, ME Altug, I Melek et al. Neuroprotective and anti-inflammatory effects of diltiazem There was no significant difference among groups with respect to mean body weight at the start of the study, or in rectal temperature, heart rate and mean arterial blood pressure values at baseline, after 20 min of ischaemia, or 5 min and 3 h after reperfusion (Table 1).

NEUROLOGICAL ASSESSMENT Neurological assessment results are summarized in Table 2. All rabbits in the sham group remained normal (Tarlov score of 5) throughout the observation period. The mean Tarlov score of rabbits in the control group was 0.66 ± 0.51, indicating severe neurological deficit of the lower extremities 24 h after ischaemia. In contrast, the mean Tarlov score of diltiazem-treated rabbits was 2.16 ± 0.75, indicating significantly improved hindlimb motor function compared with the control group (P < 0.005).

HISTOPATHOLOGICAL EXAMINATION Examination of haematoxylin and eosinstained sections of lumbar cords from animals in the sham group revealed no evidence of spinal cord injury in the white or grey matter. In the control group there was deterioration of myelinated axons in the white matter and vacuolar degeneration, eosinophilic degeneration and frank necrosis with mild inflammatory cell infiltration in the grey matter (Fig. 1). Rabbits treated with diltiazem showed only slight changes, comprising enlargement of vacuoles of glial cells in the white matter and mild vacuolization in the grey matter (Fig. 2). The quantitative and qualitative histopathological findings are shown in Table 3. Compared with the control group, the diltiazem group showed a significantly higher histopathological score (P < 0.01) and a significantly higher neuronal viability index (P < 0.001).

TABLE 1: Physiological parameters of rabbits before (baseline) and after ischaemia–reperfusion according to group randomization (mean ± SD) Variable Weight at baseline (kg) Heart rate (beats/min) Baseline Ischaemia, 20 min 5 min after reperfusion 3 h after reperfusion Mean arterial blood pressure (mmHg) Baseline Ischaemia, 20 min 5 min after reperfusion 3 h after reperfusion Rectal temperature (°C) Baseline Ischaemia, 20 min 5 min after reperfusion 3 h after reperfusion

Diltiazem (n = 6)

Control (n = 6)

Sham (n = 4)

2.2 ± 0.4

2.1 ± 0.5

2.1 ± 0.2

179 187 193 195

± ± ± ±

29 13 17 30

190 207 182 184

± ± ± ±

39 16 34 38

183 ± 34 – 184 ± 24 191 ± 34

109 88 93 98

± ± ± ±

17 10 14 11

107 89 80 94

± ± ± ±

16 18 7 13

111 ± 16 – 95 ± 9 102 ± 9

37.8 37.4 37.6 37.9

± ± ± ±

0.4 0.5 0.4 0.4

37.9 37.6 37.6 37.8

± ± ± ±

0.3 0.3 0.4 0.5

37.9 ± 0.5 – 37.8 ± 0.2 37.7 ± 0.3

No statistically significant between-group differences (P > 0.05).

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TABLE 2: Number of rabbits with various Tarlov scores and the overall scores 24 h after ischaemia–reperfusion operation according to group randomization Tarlov scorea 0 1 2 3 4 5 Overall (mean ± SD)

Diltiazem (n = 6)

Control (n = 6)

Sham (n = 4)

– 1 3 2 – – 2.16 ± 0.75b

2 4 – – – – 0.66 ± 0.51

– – – – – 4 5

a

Tarlov score: 0, spastic paraplegia with no movement of the hind limbs; 1, spastic paraplegia with slight movement of the hind limbs; 2, good movement of the hind limbs but unable to stand; 3, able to stand, but unable to hop; 4, able to hop but wobbly and might fall on their side occasionally; 5, complete recovery, able to hop normally. b P < 0.005 compared with control.

FIGURE 1: Representative photomicrograph of a lumbar spinal cord section from the control group of rabbits following surgical ischaemia–reperfusion without diltiazem treatment and sacrifice 24 h later, showing pronounced vacuolization of white and grey matter with mild mononuclear cell infiltration and neuronal degeneration (haematoxylin and eosin stain)

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FIGURE 2: Representative photomicrograph of a lumbar spinal cord section from the diltiazem group of rabbits infused with diltiazem 2 µg/kg per min starting 10 min before surgical ischaemia–reperfusion and sacrifice 24 h later, showing mild vacuolization of white and grey matter with normal neuronal structure indicated by the presence of Nissl substance in the cytoplasm, loose chromatin and prominent nucleoli (haematoxylin and eosin stain)

PLASMA IL-6 AND IL-10 CONCENTRATIONS Mean plasma IL-6 and IL-10 levels are summarized in Table 4. Sham-operated

rabbits did not show significantly different systemic IL-6 levels, a marker of the severity of systemic inflammation, compared with either control or diltiazem groups at any

TABLE 3: Quantitative and qualitative histopathological analysis of rabbit spinal cord sections 24 h after ischaemia–reperfusion according to group randomization (mean ± SD)

Neuronal viability index (quantitative)a Histopathological score (qualitative)b

Diltiazem (n = 6)

Control (n = 6)

Sham (n = 4)

0.73 ± 0.10c 3.33 ± 1.03d

0.23 ± 0.08 1.66 ± 0.81

1 4

aNeuronal

viability index: number of clearly viable neurons divided by the total neuronal count within the entire microscopic section for each animal.27 b Histolopathological score: 0, frank necrosis; 1 severe cellular damage; 2 moderate cellular damage; 3 mild cellular damage; 4 normal histological appearance.28 cP < 0.001, dP < 0.01 compared with control group.

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TABLE 4: Plasma levels of interleukin (IL)-6 and IL-10 in rabbits before (baseline) and at various times after ischaemia–reperfusion according to group randomization (mean ± SD)

IL-6 (pg/ml) Diltiazem Control Sham IL-10 (pg/ml) Diltiazem Control Sham a

Baseline

5 min after reperfusion

3 h after reperfusion

24 h after reperfusion

76.4 ± 24.4 80.3 ± 17.3 77.7 ± 11.7

77.7 ± 31.2 77.7 ± 28.2 63.1 ± 6.3

57.3 ± 25.6b 143.5 ± 39.4 92.5 ± 18.5

80.5 ± 24.8a 180.5 ± 76.9 103.3 ± 16.2

4.4 ± 1.7 5.9 ± 2.2 3.5 ± 0.8

5.4 ± 2.0 6.2 ± 1.2 4.3 ± 1.9

7.3 ± 1.8c 5.2 ± 1.4 2.7 ± 1.1

7.4 ± 1.6a,c 4.2 ± 1.9 2.4 ± 0.4

P < 0.05, bP < 0.005 compared with control group; cP < 0.005 compared with sham group.

time point. Comparison of the groups’ mean baseline IL-6 levels with measurements made 5 min, 3 h and 24 h after reperfusion also revealed no significant within group differences over time. Post hoc analysis revealed that diltiazem infusion significantly reduced mean IL-6 levels after 3 h (P < 0.005) and 24 h (P < 0.05) of reperfusion compared with the control group. Comparison of the groups’ mean baseline levels of IL-10, an anti-inflammatory cytokine that limits the effects of proinflammatory cytokines, with measurements made 5 min, 3 h and 24 h after reperfusion revealed no significant differences. By 3 h and 24 h after reperfusion, significant increases were seen in plasma IL-10 levels in the diltiazem group compared with the

sham-operated rabbits (P < 0.005). Post hoc analysis also revealed that the mean IL-10 level in the diltiazem group was significantly higher than in the control group 24 h after reperfusion (P < 0.05)

SPINAL TISSUE MDA, NO AND GSH MEASUREMENTS There were no significant differences between the groups with respect to mean spinal tissue MDA, NO and GSH measurements 24 h after ischaemia– reperfusion (Table 5).

Discussion This study provides the first evidence that diltiazem improves neurological and histopathological outcomes in a rabbit

TABLE 5: Levels of malondialdehyde, nitric oxide and reduced glutathione in spinal tissue of rabbits 24 h after ischaemia–reperfusion according to group randomization (mean ± SD)

Malondialdehyde (µmol/g protein) Nitric oxide (µmol/g protein) Reduced glutathione (mg/g protein)

Diltiazem (n = 6)

Control (n = 6)

Sham (n = 4)

54.3 ± 14.1 10.7 ± 4.2 13.0 ± 1.5

63.0 ± 10.0 15.3 ± 7.32 12.1 ± 1.9

45.4 ± 27.8 8.4 ± 0.8 14.1 ± 2.2

No statistically significant between-group differences (P > 0.05).

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I Fansa, ME Altug, I Melek et al. Neuroprotective and anti-inflammatory effects of diltiazem model of spinal cord I/R injury induced by aortic occlusion. This protection was induced by a 60-min infusion of diltiazem at 2 µg/kg per min which commenced 10 min before ischaemic insult. Ischaemia for 30 min caused by infrarenal aortic occlusion resulted in hind-limb paralysis and there was no improvement in function over 24 h in the control animals. Treatment with diltiazem improved function dramatically, as five of six rabbits were able to move their hind legs well, although they were unable to hop. The quantitative and qualitative histopathological findings, comprising neuronal viability index and histopathological scores, were consistent with the improvement in neurological outcome observed in animals that received diltiazem infusion. Haematoxylin and eosinstained tissue sections of lumbar spinal cord revealed preservation of neuronal structure, as indicated by the presence of Nissl substance in the cytoplasm, loose chromatin and prominent nucleoli 24 h after I/R injury in animals receiving diltiazem. In contrast, histological sections demonstrated eosinophilic cytoplasm, vacuolization and loss of nuclear structure, indicating severe neuronal damage and necrosis in the control group. Several experimental models of spinal cord I/R injury have been described in animals such as dogs,7 mice,29 rats,12,30 – 32 pigs11,33 and rabbits.8 – 10,27,28,34 – 44 A rabbit model for spinal cord ischaemia was used in the present study because of the unique segmental blood supply to the spinal cord from the infrarenal aorta in this animal.39 In the rabbit, aortic occlusion is a highly reliable model for the production of spinal cord ischaemia when there is a need for systematic and rapid observation of the effects of agents on spinal cord I/R injury. Rabbit spinal cord is susceptible to ischaemic

injury after infrarenal aortic occlusion because the lumbar and sacral portions receive most of their blood from segmental vessels that originate from the abdominal aorta, with poor collateral flow between the segments. The rabbit model involving aortic occlusion is a well-established spinal cord I/R injury model because there is a clear relationship between the time of occlusion, histopathological changes and resulting clinical levels of function27,28,34 – 44 This model has been used to test the potential neuroprotective effect of various agents and interventions. There is no consensus on the duration of aortic occlusion required to induce spinal cord ischaemia in the rabbit model, however the threshold of ischaemic neuronal injury has been reported as 13 – 45 min.28,34,36 – 41,43 In some studies using rabbit spinal cord ischaemia models, some rabbits could sit up or hop after 20 – 25 min of spinal cord ischaemia.40,41 However, in the present study, occlusion of the infrarenal aorta for 30 min resulted in severe neurological deficit (Tarlov scores 0 or 1) 24 h after reperfusion in all rabbits in the control group. Methods used to induce spinal ischaemia have also varied in rabbit models of spinal cord I/R injury. In some studies the aorta was occluded by inflation of an intravascular balloon catheter inserted through an arteriotomy in the femoral artery,38,41,42,44 whereas in others it was occluded with a snare or clamp using a transperitoneal approach.27,28,34,37,39,40,43 It was decided to use laparotomy and surgical anaesthesia for the aortic occlusion procedure in the present study to mimic the situation of thoracoabdominal aortic surgery. In the rabbit model used in the present study, aortic occlusion also produced ischaemia of the lower extremities. Accordingly, both the spinal cord and the

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I Fansa, ME Altug, I Melek et al. Neuroprotective and anti-inflammatory effects of diltiazem peripheral nerves were subjected to ischaemic injury. In this situation, the neurological injury induced by aortic occlusion may be a consequence of peripheral nerve injury rather than spinal cord injury. However, de Haan et al.44 evaluated the effects of peripheral ischaemia (without spinal ischaemia) on neurological deficits in rabbits. When the lower extremity was subjected to acute ischaemia for 30 min, there was no significant decrease in the amplitude of the myogenic motor-evoked response, which reflects the function of peripheral nerves and muscles. Accordingly, we believe it is unlikely that the neurological hind-limb dysfunction observed after aortic occlusion in the present study was a consequence of peripheral nerve ischaemic injury. Spinal cord injury after I/R results from the interaction of complex pathophysiological processes, such as glutamate receptormediated excitotoxicity, generation of reactive oxygen species, calcium overload, loss of membrane potential, mitochondrial failure, inflammation and apoptosis.27 – 30,34,35 – 47 Although the precise mechanism leading to spinal cord injury under I/R conditions is not completely understood, calcium (the universal second messenger) plays a central role in I/R injury.3,4,31,45,48 During reperfusion, free radical-induced membrane lipid peroxidation alters intracellular calcium homeostasis. Intracellular calcium overload is thought to initiate a series of cytoplasmic and nuclear events that lead to the activation of proteases and endonucleases, mitochondrial damage, acidosis and free radical production, and this promotes neuronal damage, apoptosis or necrosis and subsequently neurological dysfunction.3 – 5,31,48 Intracellular calcium overload of spinal cord neurons during I/R is mainly due to activation of intracellular

calcium stores and reduced reuptake or increased influx of extracellular calcium after activation of voltage-dependent calcium channels.3 – 5 These findings led us to the hypothesis that, as a voltage-dependent calcium channel blocker, diltiazem would provide neurological protection during I/R of the spinal cord. In one study, restoring blood flow during the first 10 min of reperfusion by controlling perfusion pressure to 45 – 55 mmHg exerted neuroprotective effects on the spinal cord with respect to I/R injury in the rabbit model.41 Diltiazem has modest effects on central haemodynamics compared with other calcium channel antagonists, such as verapamil and nifedipine.49 – 51 In the present study, infusion of diltiazem at 2 µg/kg per min did not lead to any significant difference in the time course of central haemodynamics, including heart rate and mean arterial pressure, compared with the control and sham groups. This suggests that mechanisms other than haemodynamic effects account for the neuroprotective effect of diltiazem against I/R injury. In the present study, we were particularly interested in the effects of diltiazem on histopathological changes and neurological recovery in temporary induced spinal cord ischaemia in rabbits. To our knowledge, there has been no formal study of the effects of diltiazem treatment on spinal cord I/R injury. The protective effect of diltiazem infusion on the spinal cord in the present study may be explained by numerous studies that have examined the effects of diltiazem on I/R injury in non-neuronal organ models.13 – 19 Virtually all knowledge about the effects of diltiazem on I/R injury comes from studies on animal hearts13 – 18 or livers.19,52,53 The beneficial effects of diltiazem on ischaemic myocardium and

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I Fansa, ME Altug, I Melek et al. Neuroprotective and anti-inflammatory effects of diltiazem liver are generally thought to be due to a vasodilatory action, which increases regional blood flow and facilitates increased microcirculation, however diltiazem might exert some action other than a haemodynamic effect. It has been shown, for example, that intracoronary diltiazem administration during early reperfusion reduced infarct size in pig hearts.18 An ultrastructural study in I/R-induced rat heart injury indicated that diltiazem prevented intracellular calcium overload through its effect on the slow L-type channel.15 Diltiazem also protects mitochondrial integrity and function, and preserves highenergy phosphates in the hearts of rabbits subjected to I/R.13,14 Some data suggest that diltiazem protects the lipid fraction within the cell membrane against the toxic effects of free oxygen radicals and, therefore, preserves membrane permeability.16,17 Additionally, diltiazem has been shown to have a protective effect by maintaining low calcium levels in hepatocytes in liver I/R injury.19,53 An in vitro study demonstrated that diltiazem reduced apoptosis in cultured rat hepatocytes subjected to I/R.53 Finally, there is also evidence that diltiazem has anti-inflammatory actions.20,21 The neuroprotective effects of diltiazem in the present study can be attributed primarily to the prevention of cytosolic calcium overload. Moreover, the antioxidant properties of diltiazem, including protection against lipid peroxidation and inhibition of the toxic effects of oxygen-derived free radicals, may be additional beneficial actions of the drug in I/R injury. Other than these protective effects of diltiazem against I/R injury, there is little information about the antiinflammatory action of diltiazem. There is increasing evidence that inflammation after reperfusion contributes to ongoing damage in spinal cord I/R

injury.32,36 – 38,54 After ischaemic insult and injury to neurons, activated leucocytes and endothelial cells release inflammatory cytokines. To date, experiments evaluating the cytokine response to spinal cord I/R have focused on tumour necrosis factor, IL-1 and IL-8.32,36 – 38 Another important aspect of the present study was the beneficial antiinflammatory effect of diltiazem, tested by measuring the levels of a proinflammatory cytokine, IL-6, and an anti-inflammatory mediator, IL-10, which inhibits the release of proinflammatory cytokines.55 To our knowledge, no formal study has evaluated how diltiazem affects inflammatory cytokine concentrations in experimental spinal cord I/R injury. Diltiazem has been shown to inhibit the release of IL-6 and stimulate the production of anti-inflammatory IL-10 at the end of cardiopulmonary bypass in patients undergoing coronary artery bypass grafting.20 The physiological significance of increasing IL-10 production was shown by Dubey and Hesong,21 who reported reduced IL-10 levels in patients with unstable coronary artery disease but significantly increased levels after diltiazem treatment in patients with unstable angina. The present study showed that diltiazem reduced IL-6 levels and stimulated IL-10 release after reperfusion. Thus, our results may provide indirect evidence of an anti-inflammatory effect of diltiazem on spinal cord I/R injury. The present study has several limitations. First, we did not record whether our I/R rabbit model had calcium overload, however, the close relationship between calcium overload and spinal cord I/R injury is well established.3 – 5 Calcium was shown to accumulate during and after ischaemia lasting > 20 min, which correlated with irreversible metabolic and functional impairment.6 It is unclear whether the protection achieved in the present study against ischaemia-induced

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I Fansa, ME Altug, I Melek et al. Neuroprotective and anti-inflammatory effects of diltiazem damage was related to the effects of diltiazem on cellular calcium increase or to some other effect, such as an increase in microcirculation during transient ischaemia. Secondly, only one calcium channel antagonist, diltiazem, was studied and only one dose was used. Thirdly, neurological function was only evaluated until 24 h after reperfusion and there was no assessment of deterioration in neurological status thereafter; some reports have shown delayed onset of neurological deficit after transient spinal cord ischaemia.33 The duration of reperfusion was 24 h in the present study, which is similar to that in previous studies of spinal cord ischaemia in rabbits.36,37,40,42 These experimental results showed that 24 h of reperfusion is sufficient for the evaluation of neurological deficit of the lower limbs and for correlating histological findings with neurological improvement. Nevertheless, it is possible that diltiazem might have a transient neuroprotective effect or it might have no beneficial effect on the occurrence of delayed paraplegia. In conclusion, the infusion of diltiazem

was shown in the present study to reduce neurological injury in a rabbit model of spinal cord I/R injury induced by aortic occlusion. This finding emphasizes that the possible neuroprotective effects of diltiazem are mediated not only by cytoprotective actions, such as preventing intracellular calcium overload, but also by its antiinflammatory properties in I/R injury. Further investigation is needed to establish whether the protective action of diltiazem is related primarily to haemodynamic factors or whether it involves metabolic protection, such as anti-inflammatory properties and the prevention of intracellular calcium accumulation.

Acknowledgement We thank Changiz Geula, Professor of Neuroscience at Northwestern University, Chicago, IL, USA, for critical reading of the manuscript.

Conflicts of interest The authors had no conflicts of interest to declare in relation to this article.

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Author’s address for correspondence Assistant Professor Iyad Fansa Mustafa Kemal University, Department of Cardiovascular Surgery, Antakya/Hatay, Turkey E-mail: [email protected]

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