Mol Neurobiol (2017) 54:8140–8150 DOI 10.1007/s12035-016-0280-x Neuroprotection by Chlorpromazine and Promethazine in Severe Transient and Permanent Ischemic Stroke Xiaokun Geng1,2 & Fengwu Li1 & James Yip2 & Changya Peng2 & Omar Elmadhoun2 & Jiamei Shen1 & Xunming Ji1,3 & Yuchuan Ding1,2 Received: 29 June 2016 /Accepted: 31 October 2016 /Published online: 28 November 2016 # Springer Science+Business Media New York 2016 Abstract Previous studies have demonstrated depressive or enhance C + P-induced neuroprotection. C + P therapy im- hibernation-like roles of phenothiazine neuroleptics [com- proved brain metabolism as determined by increased ATP bined chlorpromazine and promethazine (C + P)] in brain levels and NADH activity, as well as decreased ROS produc- activity. This ischemic stroke study aimed to establish neuro- tion. These therapeutic effects were associated with alterations protection by reducing oxidative stress and improving brain in PKC-δ and Akt protein expression. C + P treatments con- metabolism with post-ischemic C + P administration. ferred neuroprotection in severe stroke models by suppressing Sprague-Dawley rats were subjected to transient (2 or 4 h) the damaging cascade of metabolic events, most likely inde- middle cerebral artery occlusion (MCAO) followed by 6 or pendent of drug-induced hypothermia. These findings further 24 h reperfusion, or permanent (28 h) MCAO without reper- prove the clinical potential for C + P treatment and may direct fusion. At 2 h after ischemia onset, rats received either an us closer towards the development of an efficacious neuropro- intraperitoneal (IP) injection of saline or two doses of C + P. tective therapy. Body temperatures, brain infarct volumes, and neurological deficits were examined. Oxidative metabolism and stress were Keywords Hibernation-like therapeutic effect . Ischemia/ determined by levels of ATP, NADH, and reactive oxygen reperfusion . Brain metabolism . ROS species (ROS). Protein kinase C-δ (PKC-δ) and Akt expres- sion were determined by Western blotting. C + P administra- tion induced a neuroprotection in both transient and perma- Introduction nent ischemia models evidenced by significant reduction in infarct volumes and neurological deficits post-stroke. C + P Stroke is one of the most debilitating vascular diseases world- induced a dose-dependent reduction in body temperature as wide with accompanying health care costs as high as $38.6 early as 5 min post-ischemia and lasted up to 12 h. However, billion each year in the USA [1]. Reperfusion strategies such reduction in body temperature either only slightly or did not as systemic thrombolysis with intravenous (IV) tissue plas- minogen activator (tPA) and in situ clot retrieval, but not neu- roprotection strategies, remain the major therapy for stroke * Xunming Ji patients. Given the several limitations and potential complica- [email protected] tions of reperfusion therapy, however, the vast majority of * Yuchuan Ding patients with acute ischemic stroke have not benefited from [email protected] this strategy. Although a small portion of patients (17%) ex- perience spontaneous thrombolysis by 6–8h[2], many pa- 1 China-America Institute of Neuroscience, Beijing Luhe Hospital, tients suffer from permanent arterial occlusion [3]. Even if Capital Medical University, Beijing 101100, China recanalization is successful, outcome is sometimes poor due 2 Department of Neurosurgery, Wayne State University School of to reperfusion injury [4]. Medicine, 550 E Canfield, Detroit, MI 48201, USA Experimental animals have been shown to be protected 3 Department of Neurosurgery, Xuanwu Hospital, Capital Medical from the adverse effects of blood loss and oxygen deprivation University, Beijing, China when they are maintained in a suspended [5]orhibernation Mol Neurobiol (2017) 54:8140–8150 8141 state, a result of the downregulation of energy metabolism [6, maintained at physiological levels (rectal temperature at 36.5– 7]. Thus, inducing a “hibernation-like” state with depressed 37.5 °C) and one without body temperature control. Because energy utilization through the use of anesthetics has drawn of the close correlation between body and brain temperatures much interest as a potential neuroprotective strategy. [20] and increased risk of intracranial hemorrhage, rectal tem- However, limitations such as the need to apply anesthetics perature was monitored instead of brain temperature. During before the onset of ischemia as well as several toxicity issues the recovery period (24–28 h), in all temperature-controlled have narrowed their clinical potential [8]. Alternatively, hypo- groups, rats were placed on a 37 °C insulation blanket as well thermia has been recognized as a robust “hibernation-like” as under a warm light to maintain their temperature. In groups neuroprotectant because of its profoundly depressive effect without temperature control, rats were placed in a 25 °C en- on metabolism [9]. The application of hypothermia in ische- vironment. For the 4 h MCAO, rats in a total of 12 groups mic stroke patients is largely limited by the delayed cooling were randomly assigned to receive one of 3 different treat- onset, prolonged duration, extensive medical and nursing ef- ments: (1) saline, (2) 8 mg/kg C + P at 2 h after the onset of forts, and secondary complications [10]. Instead, phenothia- MCAO followed by 2.6 mg/kg (1/3 of the initial amount) 2 h zine drugs, in addition to their antipsychotic and sedative ef- later ± body temperature preservation, or (3) 12 mg/kg C + P fects, which have been demonstrated to induce “artificial hi- at2haftertheonsetofMCAOfollowedby4mg/kg2hlater bernation” [11] and neuroprotection by previous experimental without temperature control. Animals with transient MCAO work in ischemia [12–15], are therefore the focus of this study. were analyzed at 24 h of reperfusion for infarct volume, and at Since an early reperfusion strategy may not be viable for 6 or 24 h of reperfusion for protein and biochemical measure- most stroke patients, and since the ischemic regions of the ments. Similarly, for permanent stroke, rats in 8 groups were brain have patent collateral circulations for effective drug de- randomly assigned to receive one of 3 treatments: (1) saline; livery [16–18], we determined whether chlorpromazine and (2) 12 mg/kg C + P at 2 h after the onset of MCAO, followed promethazine (C + P) therapy confers neuroprotection in se- by 4 mg/kg 2 h later; or (3) 24 mg/kg C + P at 2 h after the vere stroke by reducing brain metabolism. We aimed to apply onset of MCAO, followed by 8 mg/kg 2 h later. At 28 h after C + P therapy in more clinically relevant stroke models by ischemia onset, animals from each group were examined for inducing either longer ischemia periods (4 h) or permanent neurological deficits and processed for infarct volumes or bio- (28 h) ischemia without reperfusion. As protein kinase C-δ chemical analyses. The mortality rate was low (less than 10%) (PKC-δ) and Akt/PKB are thought to play key roles in reper- and was about equal between paired groups (with or without fusion injury [19], changes in both PKC-δ and Akt were also treatment). The death of ischemic rats in the present study was investigated to understand the mechanisms underlying neuro- caused by the operative skills and skull base hemorrhage due protection induced by C + P. Results from this study could to arterial rupture during filament insertion, rather than the provide the basis for a potential stroke therapy that is relatively ischemic time. All data were analyzed in a blind manner. easy to implement. MCA Occlusion Animals were fasted 12 h prior to the pro- cedures. Animals were anesthetized in a chamber with 1–3% Materials and Methods isoflurane along with a mixture of 70% nitrous oxide and 30% oxygen. The rats were intubated, and anesthesia was main- Subjects All experimental procedures were approved by the tained with 1% isoflurane delivered from a calibrated preci- Institutional Animal Investigation Committee of Capital sion vaporizer. Rats were subjected to a right side MCAO for Medical University in accordance with the National either 2, 4, or 28 h using the intraluminal filament model [21]. Institutes of Health (USA) guidelines for care and use of lab- Reperfusion was achieved by the withdrawal of the filament at oratory animals. A total of 272 adult male Sprague-Dawley 2or4hofMCAO.BloodpCO2,pO2,meanarterialpressure rats (280–300 g, Vital River Laboratory Animal Technology (MAP), and blood glucose were monitored throughout the Co., Ltd., Beijing, China) were randomly divided into the procedure. Heating lamps and pads were utilized to maintain following groups: (1) a sham-operated group without middle rectal temperature at 36.5–37.5 °C. cerebral artery occlusion (MCAO) (n =8),(2)2hMCAO (n =8×13),(3)4hMCAO(n =8×12),and(4)permanent Chlorpromazine and Promethazine Administration In all (28 h) MCAO (n = 8 × 9). The 2 h MCAO groups were ischemia models with 2, 4, and 28 h MCAO ± reperfusion, the randomly assigned to 13 subgroups, receiving either saline combination of chlorpromazine and promethazine (1:1) at (sham treatment) or an intraperitoneal (IP) injection of two doses of 4, 8, 12, or 24 mg/kg in 3 mL saline (as determined doses C + P (1:1, 2 mg/kg + 2 mg/kg or 4 mg/kg + 4 mg/kg); by a preliminary study to induce significant neuroprotection) the first dose at 2 h after the onset of ischemia followed by a were injected IP at 2 h after the onset of ischemia. A second second dose after another hour at 1/3 of the initial amount. injection with one third of the original dose was added 1–2h Two sets of animals were used: one with body temperature later to enhance the drugs’ effects. 8142 Mol Neurobiol (2017) 54:8140–8150 Neurological Deficit The modified scoring systems (5 and 12 at p < 0.05. Post-hoc comparison between groups was scores) proposed by Zea Longa [21] and Belayev et al.