Neuroprotection by Chlorpromazine and Promethazine in Severe Transient and Permanent Ischemic Stroke

Mol Neurobiol (2017) 54:8140–8150 DOI 10.1007/s12035-016-0280-x

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 expression were determined by Western blotting. C + P administration 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 neuroprotection 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 procedures. 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. [22] achieved using the least significant difference (LSD) method. were used to examine the severity of neurological deficits in rats before surgery, during MCAO, after 24 h reperfusion, and after 28 h MCAO without reperfusion. A 28-h time point was Results chosen in MCAO without reperfusion to coincide with the 24-h reperfusion group, accounting for the 4-h reperfusion Physiological Parameters There were no significant differ- time. The severity of brain damage and consistency in each ences in blood pH, pO2, pCO2, MAP, or blood glucose be- group are highly important in this study. After MCAO, the tween the groups. modified scoring systems (five scores) for neurological deficits were used to confirm brain injury. If the scores were 1 or Body Temperature In 2 h MCAO rats, body temperatures below, the MCA occlusions were considered unsuccessful and were significantly reduced by 1 °C after C + P administration, the rats were excluded from further studies. In our study, about with both 4 and 8 mg/kg doses achieving this value within as 10% of animals with MCAO were discarded for this reason. early as 5 min (Fig. 1). At about 2 h after administration, body temperatures reached 35.7 and 34 °C with 4 and 8 mg/kg C + Cerebral Infarct Volume After 24 h of reperfusion in 2 or 4 h P, respectively. These temperatures remained significantly low MCAO, or after 28 h MCAO without reperfusion, the brains for up to 6 h and subsequently returned to normal levels. In 4 h were resected from ischemic rats and cut into 2-mm-thick MCAO rats, 3 different doses of C + P (8, 12, and 24 mg/kg slices (brain matrix) and treated with 2, 3, 5- plus an additional one-third dose) all resulted in dose- triphenyltetrazolium chloride (TTC, Sigma, USA) for stain- dependent reductions in body temperatures by 1–2°Cwithin ing. An indirect method for calculating infarct volume was as early as 5 min after administration (Fig. 1). At about 2 h used to minimize error caused by edema [23]. We also mea- after administration, body temperatures reached their lowest sured and compared infarct size of cortex and striatum at three levels, with the three different doses yielding 35.7 °C different levels from anterior +1.00 mm to posterior −4.8 mm (8 mg/kg), 32.3 °C (12 mg/kg), and 30.5 °C (24 mg/kg). to the bregma of the brain. These temperatures remained significantly depressed for up to 12 h and returned to normal levels thereafter. In the perma- ATP Production The BioVision Apo SENSOR Assay Kit nent (28 h) MCAO without reperfusion model, again, 3 dif- (Biovision, CA) was used. Brain tissue samples containing ferent doses of C + P (8, 12, and 24 mg/kg plus an additional the frontoparietal cortex and dorsolateral striatum, which are one-third dose) resulted in a dose-dependent decrease body MCA-supplied territories, were processed as described previ- temperatures by 1–2°C(p < 0.05) within as early as 5 min ously by us [24]. of C + P administration (Fig. 1). Body temperatures reached their lowest levels at 32.0 °C (24 mg/kg) after 2 h, 34.0 °C NADH Assay The quantification kit (Biovision, CA) was (12 mg/kg) after 3 h, and 35.5 °C (8 mg/kg) after 6 h post-C + used to determine brain NADH levels as described previously P administration. These temperatures remained significantly by us [24]. reduced for up to 12 h before it returned to normal levels.

Reactive Oxygen Species Production Reactive oxygen spe- Infarct Volume With2hMCAO,whenbodytemperaturesin cies (ROS) levels in the frontoparietal cortex and dorsolateral ischemic rats were maintained at 37.0 °C, both the low striatum were detected as described previously by us [25]. (4 mg/kg) (39.4%) and high (8 mg/kg) (35.7%) doses of C+Psignificantly(p < 0.05) decreased infarct volumes as Protein Expression Western blot was used to detect protein compared to non-treatment groups (50.3 %) (Fig. 2a, d), with levels of PKC-δ and phosphorylated Akt (pAkt). Brain tissues a greater infarct volume reduction seen in the higher dose. If containing the frontoparietal cortex and dorsolateral striatum body temperature was not controlled and allowed to reach a were processed as described previously by us [24, 25] and hypothermic state after C + P administration, brain infarct incubated with primary antibodies (polyclonal rabbit anti- volumes were not significantly (albeit slightly) reduced any PKC-δ at 1:5000, Santa Cruz Biotechnology, Inc.; and poly- further with either the 4 mg/kg dose (37.9 vs. 39.4%) or the clonal rabbit anti-phospho-Akt 1:1000, Cell Signaling 8 mg/kg dose (29.8 vs. 35.7%). In the 4 h MCAO group, the Technology, Inc) at 4 °C. 8 mg/kg dose with temperature controlled at 37 °C induced a mild decrease in infarct volume (41.6 vs. 50.2%, p <0.05, Statistical Analysis (SPSS Software, Version 17, SPSS Inc) Fig. 2b, e). A greater reduction (35.4%) was obtained without All data were described as mean ± SE. Differences among temperature control, but it did not reach a significant level. multiple groups were assessed using both one-way and two- However, an additional reduction (p < 0.05) in brain infarct way analysis of variance (ANOVA) with a significance level volume was further seen with higher doses at 12 (31.6%) and Mol Neurobiol (2017) 54:8140–8150 8143

Fig. 1 Drug-induced hypothermia with C + P treatment at different doses ANOVA analyses indicated that C + P significantly induced and body temperatures in a 2hMCAO,b 4hMCAO,andc permanent neuroprotection and a dose-dependent reduction in body temperature as (28 h) MCAO groups (n = 8 per group) were measured in a time- early as within 5 min and lasting up to 12 h after stroke onset dependent manner. In both transient and permanent ischemia models,

24 mg/kg (28.8%). In the permanent stroke model (Fig. 2c, f), MCA occlusion is more likely to cause more damage in the 8 mg/kg C + P did not induce significant neuroprotection cortex. No significant difference was observed between the (51.9 or 48.3 vs. 53.1%) regardless of whether temperature temperature-controlled and hypothermic groups with the 4 was maintained at 37 °C or not. However, the higher doses at or 8 mg/kg dosage in three stroke groups, possibly due to 12 and 24 mg/kg were able to reduce (p < 0.05) infarct vol- the hypothermia-independent neuroprotective effects of these umes to 44.9 and 38.2%, respectively. In addition to the total drugs taking action. infarct volume, we further determined cortical and subcortical infarct size, as well as the distribution of the infarction at Neurological Deficits Neurological deficits in the 2 h MCAO different bregma levels (Table 1). The data indicated that 4 h group followed by 24 h of reperfusion were determined by the

Fig. 2 Infarct volume reduction by dose-dependent neuroprotection and P ± temperature control after d 2h,e 4h,andf permanent (28 h) drug-induced hypothermia from C + P. TTC histology demonstrating MCAO. Treatments without temperature control were allowed to reach infarct volume reduction in the penumbra region of ischemic territory drug-induced hypothermia as indicated in Fig. 1 and demonstrated supplied by MCA with C + P ± temperature control after a 2hMCAO, slightly greater but insignificant infarct volume reduction than subjects b 4hMCAO,andc permanent (28 h) MCAO (n = 8 per group). maintained at 37 °C. #p <0.05 Percentage of infarct volume reduction (mean ± SE) with C + 8144 Mol Neurobiol (2017) 54:8140–8150

Table 1 Infarct size at three levels from anterior to posterior to 2h Level 1 Level 2 Level 3 the bregma of the brain ischemia (mean ± SE%) groups Cortex Striatum Cortex Striatum Cortex Striatum

Stroke 42.63 ± 2.10 24.96 ± 1.49 42.41 ± 2.62 24.41 ± 0.55 12.17 ± 2.79 6.48 ± 2.72 8mgtemp 35.95 ± 1.80 12.49 ± 1.22 33.57 ± 2.19 2.26 ± 0.63 10.73 ± 0.48 1.99 ± 0.18 control 8mgno 30.08 ± 2.57 11.81 ± 1.20 28.93 ± 2.82 7.81 ± 1.30 7.46 ± 1.26 1.81 ± 0.51 temp control 4h Level 1 Level 2 Level 3 ischemia Cortex Striatum Cortex Striatum Cortex Striatum groups Stroke 46.25 ± 1.20 17.73 ± 1.37 44.17 ± 1.04 28.57 ± 2.09 17.33 ± 2.90 2.95 ± 0.98 8mgtemp 38.32 ± 3.03 11.21 ± 1.31 38.36 ± 1.78 11.92 ± 1.34 9.17 ± 0.52 4.40 ± 0.78 control 8mgno 36.42 ± 2.84 9.95 ± 1.31 24.58 ± 1.53 2.98 ± 0.57 6.48 ± 1.38 0.89 ± 0.11 temp control

score systems of 5 (Fig. 3a) or 12 (Fig. 3b). Compared to no significantly (p < 0.05) reduced neurological deficits in the treatment stroke groups, neurological deficits decreased sig- 12 score system. The low temperature only enhanced the ben- nificantly (p < 0.01) after either 4 or 8 mg/kg C + P. A signif- eficial effect induced by 4 but not 8 mg/kg C + P (p <0.05) icant (p < 0.05) decrease in five score deficits was induced by (Fig. 3b). The results suggest that reduced temperature only 8 mg/kg C + P, but not by 4 mg/kg C + P. The beneficial effect play a small role in low doses of C + P, and the neurological of 4 but not 8 mg/kg was enhanced when temperature was not benefits seen here might have been due to hypothermia- controlled (Fig. 3a). C + P therapy at 4 or 8 mg/kg independent neuroprotective effects of these drugs.

ATP Levels Rats in the 2 h MCAO group showed a significant (p < 0.01) decrease in ATP levels at 6 and 24 h of reperfusion (Fig. 4a), suggesting decreased energy production or increased energy consumption. C + P at 8 mg/significantly (p < 0.01) reversed this reduction towards pre-ischemic levels, with the hypothermic effect of these drugs playing only a minor role in this reversal (p <0.05).MCAOfor4hgreatly reduced ATP levels at both 6 and 24 h reperfusion (Fig. 4b). C + P therapy at doses of 12 or 24 mg/kg attenuated (p <0.01) this reduction. In the permanent stroke model (Fig. 4c), reduced ATP levels were reversed significantly by 12 or 24 mg/kg C + P. No significant difference was found between the two doses in 4 h MCAO and permanent MCAO models.

NADH Activity Nicotinamide adenine dinucleotide (NAD+) is involved in metabolic redox reactions by transferring electrons from one reaction partner to another. Its reduced form (NADH), which donates electrons, is generated during glycolysis and through the tricarboxylic acid (TCA) cycle and ulti- mately utilized in oxidative phosphorylation to efficiently pro- Fig. 3 Neurological deficit after C + P therapy ± body temperature duce ATP in the mitochondria. In this study, cellular NADH controlin2hMCAO(n = 8 per group), using the 5 score system (a) levels, in addition to ATP production, were measured to ana- and 12 score system (b). ANOVA analyses indicated that C + P therapy p lyze the metabolic state of the ischemic brain. Compared to significantly (# < 0.05) reduced neurological deficits. Treatments p without temperature control in general had slightly but not significantly control, NADH activity was largely ( < 0.01) reduced in all better outcomes in neuroprotection ischemic rats (Fig. 5). In the 2 h MCAO model (Fig. 5a), C + P Mol Neurobiol (2017) 54:8140–8150 8145

Fig. 4 Improvement in ATPlevels after C + P therapy (n =8pergroup).a Decrease in ATP levels was seen after 6 and 24 h of reperfusion after 2 h MCAO (##p < 0.01). Reestablishment of ATP levels to baseline was induced (*p < 0.05) by 8 mg/kg C + P and was not significantly Fig. 5 Improvement in NADH levels after C + P therapy. a Decrease in p enhanced by low temperature. b Dose-dependent reestablishment of NADH levels was seen (## < 0.01) after 6 and 24 h of reperfusion in the n ATP levels to baseline after 12 or 24 mg/kg C + P in the 4 h MCAO 2 h MCAO groups ( = 8 per group). NADH levels reestablished by group at 6 and 24 h of reperfusion (##p < 0.01 between control and no 8 mg/kg C + P were not further improved by drug-induced treatment,**p < 0.01 between 12 mg/kg C + P and no treatment, hypothermia. b ANOVA analyses indicated that reduced NADH levels p **p < 0.01 between 24 mg/kg C + P and no treatment). c Reduction (## < 0.01) in the 4 h MCAO group after 6 and 24 h of reperfusion were p p (##p < 0.01) in ATP levels seen after permanent stroke (28 h MCAO reestablished by 12 or 24 mg/kg C + P (* <0.05,** <0.01).c Again, p without reperfusion) was reestablished (**p < 0.01) by 12 or 24 mg/kg reduced NADH levels (## < 0.01) and its reestablishment by 12 or p C+P 24 mg/kg C + P (* < 0.05) was observed in the permanent (28 h) MCAO without reperfusion group therapy at 8 mg/kg reversed NADH activity towards pre- ischemic levels at 6 h of reperfusion, while temperature did (Fig. 6a). Body temperature did not play a large role in this not affect NADH levels. After 4 h MCAO (Fig. 5b) or perma- reduction. In tandem administration of 12 or 24 mg/kg doses nent stroke (Fig. 5c), NADH activity was significantly re- of C + P significantly (p < 0.01) reduced ROS levels in the 4 h versed by either 12 or 24 mg/kg C + P at both 6 and 24 h of (Fig. 6b) and permanent (Fig. 6c) MCAO groups. While both reperfusion. Again, no significant difference was found be- the 12 and 24 mg/kg C + P doses significantly reduced ROS tween the two doses. levels towards pre-ischemic levels, there was no significant difference between the two doses. Oxidative Stress Compared to the sham-operated group, ischemia for 2, 4, or 28 h (permanent) significantly (p <0.01) Akt and PKC-δ Protein Compared to sham control (refer- increased ROS production (Fig. 6). In 2 h MCAO groups enced as 1, not shown), there was a significant decrease followed by either 6 or 24 h of reperfusion, C + P therapy at (p < 0.01) in pAkt levels after 2 h MCAO at both 6 (Fig. 7a) 8 mg/kg significantly (p < 0.01) attenuated this increase and 24 h (Fig. 7b) of reperfusion. C + P treatment increased 8146 Mol Neurobiol (2017) 54:8140–8150

Fig. 7 Increase in pAkt and decrease in PKC-δ levels seen after C + P therapy after 2 h MCAO (n = 8 per group). Compared to sham control (referenced as 1, not shown), ANOVAanalyses indicated that there was a significant decrease (p < 0.01) in pAkt levels after stroke at both a 6andb 24 h of reperfusion. Ischemia-induced reduction in pAkt levels was significantly reversed (**p < 0.01) by C + P treatments, with 8 mg/kg being more effective than the 4 mg/kg dose. Furthermore, there was a significant increase in PKC-δ levels at a 6 and b 24 h (p < 0.01) of reperfusion. Both doses of C + P reversed ischemia-induced PKC-δ elevation, with the 8 mg/kg dose exhibiting the greatest reduction at 6 n Fig. 6 Reduction in ROS production after C + P therapy ( = 8 per and 24 h (*p < 0.05) of reperfusion. Representative immunoblots are p group). a Increase in ROS levels was seen (** < 0.01) at 6 and 24 h of presented reperfusion in the 2 h MCAO group. ROS levels were reduced by 8 mg/kg C + P at both 6 and 24 h of reperfusion and further reduced only at 6 h of reperfusion when temperature was not controlled (##p <0.01).b ANOVA analyses indicated that ROS production was Discussion increased in the 4 h MCAO group after 6 and 24 h of reperfusion (*p < 0.05). Dose-dependent reduction of ROS levels was induced by Phenothiazine and Neuroprotection This present study, for p p 12 or 24 mg/kg C + P (## <0.01## <0.01).c Increase in ROS levels the first time to our knowledge, revealed a dose-dependent was seen in the permanent (28 h) MCAO group (**p < 0.01). This increase was largely reduced to baseline by 12 or 24 mg/kg C + P neuroprotective effect of phenothiazine drugs following tran- (##p < 0.01), with no significant difference between the two doses sient and permanent ischemia. Since reperfusion therapy within a clinically realistic time window is not available to most stroke patients, a goal of the study was to develop a viable neuroprotective therapy by assessing the beneficial effects of (p < 0.01) pAkt levels at both time points, with the 8 mg/kg C + P in severe ischemic conditions (4 and 28 h occlusion), in dose producing a better neuroprotective effect than the contrast to those used in a previous study with moderate stroke 4 mg/kg dose at 6 h of reperfusion. Conversely, there was a periods (2 h occlusion or less) [23, 26–28]. Although C + P significant increase in PKC-δ levels at 6 (p <0.01;Fig.7a) therapy also reduced body temperature, this effect had very and 24 h (p <0.01;Fig.7b) of reperfusion. Again, either of the limited therapeutic effects. These results suggest that drug- C + P doses decreased PKC-δ expression, although 8 mg/kg induced hypothermia in this case did not play significant role exhibited the greatest reduction after 6 h of reperfusion. As in neuroprotection, but instead was a result of the C + P-induced hypothermia did not largely improved post- hypothermia-independent effects of these drugs. occlusion outcomes based on previous experiments, the effect Since the 1950s, chlorpromazine and promethazine, serv- of body temperature was not determined in this analysis. ing as prototypes for the phenothiazine class of drugs, have Mol Neurobiol (2017) 54:8140–8150 8147 been widely used as neuroleptics because of their antipsychot- oxidation on the nervous system. This impaired carbohydrate ic and sedative effects [29, 30]. These two drugs, which have utilization may occur even under normal oxygen consumption been commonly used in combination [30], are two of the [48]. Moreover, the induced-hyperglycemic response could be oldest and well-studied drugs from the phenothiazine class. blocked by promethazine [49, 50]. Our parallel studies indi- Due to their high lipophilicity, C + P easily pass the blood- cated a “hibernation-like” effect of these phenothiazine agents brain barrier (BBB) to exert depressive metabolic effects on on suppressing glucose utilization and brain metabolism (data the central nervous system. Chlorpromazine is a low-potency not shown), which are similar to that of anesthetic drugs, such typical antipsychotic with primarily anticholinergic and as pentobarbitone [51, 52]. Not only does administration of antiadrenergic side effects. It has been reported to interfere phenothiazines cause a reduction in blood flow and oxygen with a number of receptors and ion channels, including sero- consumption [53], but they also reduce energy metabolism tonin receptors, Ca2+channels, K+ channels, and Cl-channels [12, 44]. [31]. Chlorpromazine has been reported to block excitatory We have previously reported a suppressive effect of alcohol glutamatergic signal transmission by inhibiting NMDA recep- on brain metabolism which leads to neuroprotection following tors [32]. It also confers neuroprotection against brain ische- the onset of ischemic stroke [23–25]. However, in comparison mia by activating Ca2+-associated potassium channels by lim- to phenothiazine drugs, there are many societal implications iting Ca2+ entry, thus reducing excessive release of excitatory and significant side effects associated with alcohol which may neurotransmitters and energy expenditure [31]. limit its clinical translation. In addition to their clinical use for Chlorpromazine has been shown to exert neuroprotective ef- their antipsychotic and sedative effects, the present experi- fect on the spinal cord ischemia [33], glutamate-induced neu- mental studies along with previous studies [12–14]supporta rotoxicity [34], ethanol-induced neuronal apoptosis [35], and new application of phenothiazine drugs as a neuroprotectant in vitro hippocampal neuronal cell death [36]. Promethazine, in stroke for their ability to induce an “artificial hibernation” on the other hand, is a first-generation antihistamine clinically status [11]. used for its strong sedative, antiemetic, and weak antipsychot- PKC-δ is upregulated in ischemic stroke and is potentiated ic effects. Promethazine has been shown to inhibit NMDA by the accumulation of ROS [19, 54, 55]. There are several receptors, Na+/K+-ATPase, and the mitochondrial permeabil- pathways in cerebral glucose metabolism that may affect ity transition pore, all of which may contribute to its sedative PKC. In hyperglycemic states, glucose can elevate diacylglyc- and neuroprotective effects [37]. Mitochondrial permeability erol (DAG), a second messenger lipid essential for PKC acti- transition pores cause leakage in the mitochondrial inner vation, or activate microvascular PKC isoforms to result in membrane and subsequently result in cell death. By this mech- vascular complications [56]. Fibroblast growth factors (FGF) anism, promethazine may reduce ischemic neuronal injury such as FGF-1 and FGF-2 may also be involved in neuropro- such as those found in stroke patients [37–41]. tection against insults such as ischemia, injury, or stress [57, Phenothiazine derivatives have been reported to protect hu- 58]. Studies have shown FGF to be mediated by Src tyrosine man cells against oxidative stress [42]. kinase phosphorylation and phospholipase Cγ, leading to increased DAG production and PKC activation [59]. FGFs were “Hibernation-Like” Effect on Brain Metabolism Energy also shown to activate PKC through SNT/FRS2 [59]. failure and oxidative stress with ROS generation, secondary Although the entire mechanism is still unclear, here, we have to impaired oxidative phosphorylation and increased anaero- shown for the first time that C + P downregulates PKC-δ,thus bic glycolysis (hyperglycolysis), are well-documented patho- making these drugs possible therapeutic strategies for future physiologies of ischemic neuronal injury [43]. Recent re- ischemic stroke studies. search, however, has failed to develop targeted therapies that Akt, also known as PKB, is a protein kinase involved in may confer neuroprotection acutely after stroke by addressing multiple pathways such as in the cell cycle and insulin signal- the above metabolic dysfunctions [43]. Current study with ing pathways. Studies have elucidated in detail that Akt carries C + P in transient or permanent ischemia demonstrated a tem- out the downstream effects controlled by a class of kinases poral relationship between early improvement in ATP and known as phosphoinositide 3-kinases (PI 3-kinases). Akt’s NADH production and reduced ROS generation with subse- involvement in insulin signaling could be another method by quent reduction in infarct volume and neurological deficits. which Akt prevents neuronal damage following stroke by Although the measures here cannot conclusively explain the promoting normal metabolic process and recovery [60]. better outcomes, our findings support a role of C + P in stabi- Although the exact mechanism of Akt-associated neuropro- lizing the dysfunctional metabolic pathways mediated by the tection still needs to be elucidated, our results show that post- PKC and Akt signaling in ischemia. ischemic Akt levels increased with C + P administration, C + P have been shown to alter glucose metabolism [44] supporting their potential in this stroke therapy. While the and inhibit glucose uptake [12, 45–47], suggesting that their primary focus here is centered on the treatment effects in is- depressing effects may involve inhibiting carbohydrate chemic brain tissues, additional mechanistic studies are 8148 Mol Neurobiol (2017) 54:8140–8150 needed to assess the correlation between functional outcomes Compliance with Ethical Standards All experimental procedures and the regulatory roles of the PKC-Akt signaling. At a more were approved by the Institutional Animal Investigation Committee of Capital Medical University in accordance with the National Institutes of fundamental level, future studies will aim to determine the Health (USA) guidelines for care and use of laboratory animals. cause-and-effect relationships between C + P outcomes and its regulators in ischemic, as well as non-ischemic, brain Sources of Funding This work was partially supported by American tissues. Heart Association Grant-in-Aid (14GRNT20460246), Merit Review Award (I01RX-001,964-01) from the US Department of Veterans Affairs Rehabilitation R&D Service, National Natural Science Foundation of China (81501141), and Beijing NOVA program Clinical Perspectives of C + P Therapy This study demon- (xx2016061) as well as National Outstanding Youth Science Fund of strated the therapeutic possibilities of C + P in a severe ische- China (no. 81325007). mic stroke model. There may be a concern that a large infarct in our model may compromise the hypothalamic arteries, Disclosures None. leading to hypothalamic infarction and consequently temperature dysregulation. However, the arterial supply of the hypo- References thalamus is derived from multiple perforating vessels from various parts of the circle of Willis and the superior hypophy- 1. 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