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Logo of jspinalcordmedThe Journal of Spinal Cord Medicine
 
J Spinal Cord Med. July, 2015; 38(4): 538–543.
PMCID: PMC4612210

Effects of adenosine monophosphate-activated kinase in the ventral horn of rabbit spinal cord after transient ischemia

Abstract

Objective

To investigate the effect compound C, an adenosine monophosphate-activated kinase (AMPK) inhibitor, has on motor neurons of rabbit spinal cord after ischemia/reperfusion.

Design

Compound C (30 mg/kg) was administered intraperitoneally to rabbits 30 minutes before ischemia and the animals were sacrificed at 15 minutes after ischemia/reperfusion to measure lactate levels and at 72 hours after ischemia/reperfusion for morphological study.

Results

The administration of compound C did not produce any significant changes in physiological parameters such as pH, arterial blood gas (PaCO2 and PaO2), and blood glucose in rabbit either at 10 minutes before ischemia or at 10 minutes after reperfusion. However, the administration of compound C did significantly ameliorate lactate acidosis at 15 minutes after reperfusion. In addition, the administration of compound C significantly improved the neurological scores of the rabbits and reduced the neuronal death seen in the ventral horn of their spinal cords at 72 hours after ischemia/reperfusion.

Conclusions

Inhibition of AMPK can ameliorate the ischemia-induced neuronal death in the spinal cord via the reduction of early lactate acidosis.

Keywords: Adenosine monophosphate-activated kinase, Transient spinal cord ischemia, Neuroprotection, Lactate, Rabbit

Introduction

Transient spinal cord ischemia can cause paraplegia as an unpredictable complication after the surgical repair of thoracic or thoracoabdominal aneurysms.1 The incidence of paraplegia induced by this surgical repair has been reported to be 3–18%.2 The rabbit is a reliable model for spinal cord ischemia because a rabbit's spinal cord blood supply is segmental,3 and recent studies have shown residual flow to the spinal cord in aortic clamping to be on the order of 2% or less.4

It has been studied that therapeutic strategy to reduce severity of ischemic spinal cord injury using various methods such as cerebrospinal fluid drainage, hypothermia, distal bypass, managing the blood pressure, and adjunctive pharmacological agents.5 In addition, numerous pharmacological agents have neuroprotective effects against ischemic damage including antioxidants, oxygen radical scavenger, and anti-inflammatory agents.610 The interruption of blood flow by aortic clamping significantly reduces the blood supply in the spinal cord and can cause neuronal damage in the ventral horn of the spinal cord because of the high levels of neuronal glucose utilization. Oxygen demand and the metabolic rate is one the most important factors in the spinal cord after ischemia. Adenosine monophosphate-activated kinase (AMPK) is one of the master sensors and regulators of energy balance, and AMPK can be activated when ATP supply does not keep pace with demand or when the ATP/AMP ratios decrease. AMPK is a heterotrimeric protein, comprised catalytic α subunits (α1, α2), and β and γ regulatory subunits (β1, β2 and γ1, γ2, γ3)11; it is activated via phosphorylation by an upstream kinase.12,13 The phosphorylation of AMPK increases ATP levels by facilitating fatty acid oxidation and decreasing cholesterol synthesis.14

Several studies have indicated that a reduction in AMPK activation leads to neuroprotection in a reversible experimental model of focal stroke.1517 In addition, inhibition of the AMPK α2 catalytic subunit in AMPK prevents motor neuron death in amyotrophic lateral sclerosis.18 There were no studies on the effect an AMPK inhibitor has against transient ischemic damage in the spinal cord. Compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine) (Sigma-Aldrich, St. Louis, MO, USA) is also known as dorsomorphin and is a small-molecule inhibitor of AMPK. Treatment of compound C to mice results in dephosphorylation and inactivation of AMPK and induces profound anorexia.19 In addition, compound C increases ATP levels in the neurons.20,21 Therefore, in this study, we investigated the effect of compound C against ischemic damage in the rabbit spinal cord after 15 minutes of transient ischemia.

Methods

Experimental animals

Twenty-four male New Zealand white rabbits (1.2–1.5 kg) were obtained from the Experimental Animal Center (Cheonan Yonam College, Cheonan, South Korea). They were housed in a conventional state under adequate temperature (21°C) and humidity (60%) controls with a 12-hour light/12-hour dark cycle and could freely access food and tap water. The handling and caring of the animals conformed to the guidelines established in order to comply with current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85–23, 1985, revised 1996) and were approved by the Institutional Animal Care and Use Committee (IACUC) of Hallym University. All of the experiments were conducted with an effort to minimize the number of animals used and the suffering caused by the procedures used in this study.

Experimental group and drug treatment

To elucidate the effects of compound C against ischemic damage, the animals were divided into two groups: sham-operated (control) and ischemia-operated group. The latter was further divided into two subgroups: vehicle (physiological saline)-treated group and 30 mg/kg compound C-treated group. The dosage of compound C was adopted because our previous study showed neuroprotective effects against ischemic damage in this dosage.22 The vehicle and compound C was intraperitoneally administered to rabbits at 30 minutes before ischemia. Arterial blood gases and blood glucose were measured using a GEM Premier 3000 gas analyzer (Instrumentation Laboratory, Milan, Italy) at 10 minutes before ischemia and 10 minutes after reperfusion, respectively.

Induction of transient spinal cord ischemia

The animals were anesthetized with a mixture of 2.5% isoflurane (Baxtor, Deerfield, IL) in 33% oxygen and 67% nitrous oxide. A ventral midline incision was made in the abdomen. The abdominal aorta was isolated underneath the left renal artery and occluded using a non-traumatic aneurysm clip to avoid the principal mesenteric flow to the gut. Thereafter, spinal cord ischemia was made according to a modified method reported by Kiyoshima et al.23 After 15 minutes of occlusion, the aneurysm clip was removed, and the reperfusion was observed from the abdominal aorta. The 15-minute clamping produced delayed paraplegia within several hours after ischemia.24 Body temperature was maintained under free-regulating or normothermic (38.7 ± 0.3°C) conditions, monitored with a rectal temperature probe (TR-100; Fine Science Tools, Foster City, CA, USA), and maintained using a thermometric blanket before, during, and after the surgery until the animals completely recovered from anesthesia. Thereafter, the animals were kept on the thermal incubator (Mirae Medical Industry, Seoul, South Korea) to maintain body temperature until the animals were euthanized. Control animals were subjected to the same surgical procedures except the abdominal aorta was not occluded.

Neurological assessment

For neurological function assessment, modified Tarlov criteria were used25: 0, no voluntary hind-limb function; 1, only perceptible joint movement; 2, active movement but unable to stand; 3, able to stand but unable to walk; or 4, complete normal hind-limb motor function.26,27 All assessments were performed to ensure objectivity in blind conditions by two observers for each experiment, carrying out assessments in the control, vehicle-treated, and compound C-treated groups (n = 5 in each group) under the same conditions. Neurological functions were assessed at 72 hours after reperfusion because the neurologic function of the animals starts to deteriorate at about 12–24 hours after the reperfusion, progressing to complete delayed-onset paraplegia within ensuing 2 days.28,29

Cresyl violet staining for cell viability in the spinal cord

For the histological analysis, the animals used in neurological assessment were anesthetized with 2 g/kg urethane (Sigma) and perfused transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4), which was followed by 4% paraformaldehyde in 0.1 M phosphate-buffer (PB, pH 7.4). The animals’ spinal cords were removed, and the fifth to sixth lumbar segments (L5–L6) of the spinal cord were postfixed in the same fixative for 4 hours. The spinal cord tissues were cryoprotected by infiltration with 30% sucrose overnight, and 30-μm-thick spinal cord sections in the coronal plane were serially cut using a cryostat (Leica, Wetzlar, Germany). The sections were stained with cresyl violet acetate as previously described.22

The measurement of cresyl violet-positive cells in all the groups was performed using an image analysis system equipped with a computer-based CCD camera (software: Optimas 6.5, CyberMetrics, Scottsdale, AZ, USA) corresponding to a tissue area under a 100× primary magnification. In brief, cresyl violet-positive neurons were counted at the center of the ventral horn of spinal cord. The image was converted to gray image and automatically selected the cresyl violet-positive neurons according to its cresyl violet-stained density. Cell counts were obtained by averaging the counts from 25 sections with 300-μm intervals from each animal.

Lactate levels in the spinal cord

To confirm the effects of compound C on lactate levels in the spinal cord, animals (n = 3) in vehicle- treated and 30 mg/kg compound C-treated groups were used in this study. At 15 minutes after reperfusion, the animals were anesthetized with 2 g/kg urethane (Sigma) and the spinal L5–L6 segments were cut to 500-μm thickness in a vibratome (Leica Microsystems, GmbH, Germany). The tissues were extracted with 20 µl 0.1 M NaOH in methanol. The extracts were moved to an ice bath, 50 µl 0.05 M NaOH was added, and the mixture was heated at 95°C for 10 minutes to inactivate tissue enzymes. The extracts were then assayed for lactate using enzymatic methods. In brief, extracts were neutralized to pH 7.4–8.0 with 7.5 N KOH/50 mM NaH2PO4, centrifuged to remove the precipitate, and the lactate concentration in the final supernatant was measured spectrophotometrically by using commercial assay kits (Sigma).

Statistical analysis

The data presented represent the means of the experiments performed for each experimental investigation. The differences among the means were statistically analyzed by a one-way analysis of variance followed by a Tukey's post hoc method in order to compare the effects of Compound C against ischemic damage.

Results

Effect of compound C on ischemia-induced hindlimb motor function

Physiological parameters such as pH, arterial blood gas (PaCO2 and PaO2), and blood glucose level did not show any significant changes after ischemia/reperfusion. In addition, no significant differences in pH, arterial blood gas (PaCO2 and PaO2), or blood glucose were observed after compound C treatment at either 10 minutes before or 10 minutes after reperfusion (Table 1).

Table 1
Physiological parameters at 10 minutes before ischemia and 10 minutes after reperfusion**

Effect of compound C on ischemia-induced hindlimb motor function

In the control group, all animals showed normal behavior and had a neurological score of 4 at 72 hours after sham operation. In the vehicle-treated group, most animals showed hemiplegia and had a neurological score of 0 or 1 at 72 hours after ischemia/reperfusion. In this group, the average neurological score was 0.8. In the compound C-treated group, most animals showed movement of the hindlimb with a neurological score of 2 or 3. In this group, the average neurological score was 2.2 (Fig. 1).

Figure 1
Neurological function scores in each animal measured by the Tarlov criteria in the control, vehicle-treated, and compound C-treated groups at 72 hours after sham or ischemia/reperfusion. The mean values of neurological function scores are also shown ( ...

Effect of compound C on motor neurons in the ventral horn of the spinal cord

In the control group, cresyl violet-positive neurons were thoroughly distributed in the ventral horn of the spinal cord at 72 hours after sham operation (Fig. 2A). In this group, the mean number of cresyl violet-positive neurons was 20.4 per section in the ventral horn of the spinal cord (Fig. 2D). In the vehicle-treated group, a few cresyl violet-positive neurons were detected in the ventral horn (Fig. 2B) and the average number of cresyl violet-positive neurons was 6.65 per section in the ventral horn (Fig. 2D). In the compound C-treated group, cresyl violet-positive neurons were abundant in the ventral horn (Fig. 2C) and the average number of cresyl violet-positive neurons was 15.3 per section in the ventral horn (Fig. 2D).

Figure 2
Staining of surviving neurons by cresyl violet in the ventral horn of the L5–L6 spinal cord in the control (A), vehicle-treated (B), and compound C-treated (C) groups at 72 hours after sham or ischemia/reperfusion. In the control group, cresyl ...

Effect of compound C on lactate acidosis in the spinal cord

In the vehicle-treated group, lactate levels were significantly increased to 182.7% of the control group at 15 minutes after reperfusion. In the compound C-treated group, lactate levels were significantly decreased compared with levels found in the vehicle-treated group and were 76.8% of levels in the vehicle-treated group (Fig. 3).

Figure 3
Levels of ATP in the L5–L6 spinal cord homogenates in the control, vehicle-treated, and compound C-treated groups at 15 minutes after sham or reperfusion (n = 3 per group; *P < 0.05 versus the control group; ...

Discussion

AMPK is activated when there is a rise in AMP levels or an increase in the AMP/ATP ratio.16 The activation of AMPK may be one of the important compensatory mechanisms that can modulate cellular energy deficiency. AMPK activation can be either beneficial or deleterious, depending on the tissue, degree of stimulation, or conditions of activation. Overexpression of active AMPK or treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) enhanced oxidative stress and induced apoptosis in mouse neuroblastoma cells through the activation of nuclear factor-κB.30 In an in vivo model of stroke, the administration of AICAR increases the injury after occlusion of the middle cerebral artery.31 In addition, AMPK activation leads to increased production of Aβ peptides, which contributes to amyloid plaque formation in Alzheimer's disease.32 In this study, we observed the effect of compound C on motor neurons in the ventral horn of the spinal cord. Compound C significantly improved neurological scores and increased neuronal survival in the ventral horn of the spinal cord at 72 hours after ischemia/reperfusion, even though pH, arterial blood gas (PaCO2 and PaO2), and blood glucose did not show any significant changes in the rabbit at either 10 minutes before ischemia or 10 minutes after reperfusion.

This, to our knowledge, is the first study to show that inhibition of AMPK protects neurons from ischemic damage in the spinal cord; however, several reports have shown that pharmacological inhibition or genetic depletion of AMPK leads to a reduction in metabolic demand, and under conditions of acute ischemia, protects the brain.1517,22,31,33

In an ischemic state, glucose is metabolized anaerobically because of the depletion of oxygen. With reperfusion, as glucose and oxygen again become available for metabolism, lactic acid may continue to be produced, temporarily accumulating in even greater quantities than during the ischemic period.34,35 It was reported that increase of lactate in the pathological region of brain and spinal cord using a proton magnetic resonance imaging.36 It was also reported that neuropathological changes and flaccid hindlimb paralysis were resulted from increased lactic acid in a cerebrospinal fluid induced by dynorphin A injection.37 In this study, we observed the lactate levels in the spinal cord at 15 minutes after reperfusion because the production of lactic acid peaks at 15 minutes after reperfusion in the rabbit spinal cord.38 The administration of compound C significantly reduced the accumulation of lactate in the spinal cord. This result was supported by that of a previous study showing that Compound C may exert neuroprotection in ischemic stroke through inhibiting lactic acidosis.16,22

Conclusion

Pharmacological inhibition of AMPK protects motor neurons from ischemic damage in the spinal cord by reducing early lactate accumulation.

Disclaimer statements

Contributors BMC and WK design this study and wrote the manuscript. SMM supervised and designed this study. DYY, HYJ conducted the morphological study. JHC, M-HW, and IKH advised the study and conduct the physiological study.

Funding This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MEST), Republic of Korea (2010-0021445).

Conflicts of interest None.

Ethics approval The handling and caring of the animals conformed to the guidelines established in order to comply with current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985, revised 1996) and were approved by the Institutional Animal Care and Use Committee (IACUC) of Hallym University.

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