Laboratory Study
Effects of citicoline used alone and in combination with mild hypothermia on apoptosis induced by focal cerebral ischemia in rats

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Abstract

The effects of citicoline used either alone or in combination with hypothermia on the suppression of apoptotic processes after transient focal cerebral ischemia were investigated. Middle cerebral artery occlusion (MCAo) was performed for 2 hours on Sprague-Dawley (SD) rats using intraluminal thread insertion. The treatment groups were as follows: Group 1, sham-operated; Group 2, saline; Group 3, citicoline (400 mg/kg intraperitoneal.); Group 4, hypothermia (34 ± 1 °C); Group 5, citicoline + hypothermia. All rats were reperfused for 24 hours, and after sacrifice and transcardiac perfusion, immunohistochemical studies were performed for markers of apoptosis. In Group 2, the Bcl-2 immunostaining score (mean ± standard deviation, 0.71 ± 0.75) was lower compared to Groups 3, 4 and 5 (2.33 ± 0.81; 3.00 ± 0.00; 2.20 ± 0.83; p < 0.05). There was higher expression of caspase-3 proteins in Group 2 (2.28 ± 0.95) compared to Group 5 (1.50 ± 0.83; p < 0.05). Bax proteins were also increased in Group 2 (1.85 ± 1.06) compared to Group 5 (0.40 ± 0.54) and in Group 4 (2.00 ± 0.00) compared to Group 5 (0.40 ± 0.54; p < 0.05). Significant differences in caspase-9 immunostaining scores were found in Group 2 (2.29 ± 0.96) compared to Group 5 (0.20 ± 0.44) (p < 0.05); Group 3 (1.00 ± 0.70) compared to Group 5 (0.20 ± 0.44; p < 0.05); and Group 4 (3.00 ± 0.00; p < 0.05) compared to Group 5 (0.40 ± 0.54; p < 0.05). Thus by suppressing apoptotic processes citicoline with hypothermia is more effective than either used alone in ameliorating cerebral damage after transient focal ischemia.

Introduction

Neuronal cell death during stroke occurs, particularly in the central zone, by necrotic and apoptotic mechanisms.1 Apoptosis following cerebral ischemia/reperfusion is a major pathway leading to cell death; the presence of apoptotic cells in the penumbra suggests that apoptosis may contribute to the final infarct size.2 Proapoptotic protein translocation during cerebral ischemia/reperfusion is controlled by the family of Bcl-2 proteins. The anti-apoptotic protein Bcl-2 has been implicated following brain injury, and is thought to provide protection after transient focal ischemia.3

In this study, we tested the effects of the combination of hypothermia, which has elicited neuroprotective effects in experimental and clinical studies,[4], [5], [6], [7] and citicoline, an endogenous compound that stabilizes membrane function and reduces free radical generation, during cerebral ischemia.[8], [9], [10] Krupinski et al.11 showed that citicoline may directly interact with the apoptotic cascade by inhibiting pro-caspase expression and caspase activation, and suggested that this effect was probably due to the interaction of citicoline with phosphatidylcholine metabolism. Study of the combination of hypothermia and citicoline on cerebral ischemia seems rational, since these agents act through different mechanisms and may therefore have a synergistic effect.

We investigated the neuroprotective effects of citicoline and mild hypothermia (34 ± 1 °C), used either alone or in combination, on apoptotic processes using the middle cerebral artery occlusion (MCAo) model. The purpose of the study was to determine whether the combination treatment showed additive benefit in reducing apoptosis.

Section snippets

Materials and methods

The experimental procedures and protocols used in this study were reviewed and approved by our Institutional Committee on Animal Research, and were carried out in accordance with its guidelines.

Results

There were no differences in body weight of the rats in any of the groups. Body temperatures (mean ± standard deviation) of Group 4 and Group 5 rats were significantly reduced due to hypothermia (Group 1: 37.08 ± 0.03; Group 2: 37.22 ± 0.01; Group 3: 36.98 ± 0.03; Group 4: 33.96 ± 0.26; Group 5: 33.95 ± 0.03) (p > 0.05).

Immediately after MCAo, LCBF measurements (mL LD/100 g per minute) decreased sharply by over 50% compared to controls. All groups exhibited significant decreases in LCBF after MCAo. The

Discussion

There is increasing evidence that some neuronal death after brain ischemia is mediated by the action of cysteine-requiring aspartate directed proteases (caspases), the proteases responsible for apoptosis in mammals.18 Caspase-mediated neuronal death is more extensive after transient than permanent focal brain ischemia, and may contribute to delayed loss of neurons in the penumbral region of infarcts. The intracellular processes that contribute to caspase-mediated neuronal death following

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