A novel stroke therapy of pharmacologically ... - The FASEB Journal

The FASEB Journal • Research Communication

A novel stroke therapy of pharmacologically induced hypothermia after focal cerebral ischemia in mice Ko-Eun Choi,* Casey L. Hall,† Jin-Mei Sun,* Ling Wei,*,† Osama Mohamad,* Thomas A. Dix,‡,§ and Shan P. Yu*,‡,1 *Department of Anesthesiology and †Department of Neurology, Emory University School of Medicine, Atlanta, Georgia, USA; ‡Department of Pharmaceutical and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina, USA; and §Halimed Pharmaceuticals, Charleston, South Carolina, USA Compelling evidence from preclinical and clinical studies has shown that mild to moderate hypothermia is neuroprotective against ischemic stroke. Clinical applications of hypothermia therapy, however, have been hindered by current methods of physical cooling, which is generally inefficient and impractical in clinical situations. In this report, we demonstrate the potential of pharmacologically induced hypothermia (PIH) by the novel neurotensin receptor 1 (NTR1) agonist ABS-201 in a focal ischemic model of adult mice. ABS-201 (1.5–2.5 mg/kg, i.p.) reduces body and brain temperature by 2–5°C in 15–30 min in a dose-dependent manner without causing shivering or altering physiological parameters. Infarct volumes at 24 h after stroke are reduced by ⬃30 – 40% when PIH therapy is initiated either immediately after stroke induction or after 30 – 60 min delay. ABS-201 treatment increases bcl-2 expression, decreases caspase-3 activation, and TUNEL-positive cells in the peri-infarct region, and suppresses autophagic cell death compared to stroke controls. The PIH therapy using ABS-201 improves recovery of sensorimotor function as tested 21 d after stroke. These results suggest that PIH induced by neurotensin analogs represented by ABS-201 are promising candidates for treatment of ischemic stroke and possibly for other ischemic or traumatic injuries. Choi, K.-E., Hall, C. L., Sun, J.-M., Wei, L., Mohamad, O., Dix, T. A., Yu, S. P. A novel stroke therapy of pharmacologically induced hypothermia after focal cerebral ischemia in mice. FASEB J. 26, 2799 –2810 (2012). www.fasebj.org ABSTRACT

Key Words: neurotensin analog 䡠 brain protection 䡠 autophagy 䡠 sensorimotor function

Abbreviations: 3-MA, 3-methyladenine; BBB, blood-brain barrier; bcl-2, B-cell lymphoma 2; CC3, cleaved caspase-3; CCA, common carotid artery; i.c.v., intracerebroventricular; i.p., intraperitoneal; i.v., intravenous; LC3, microtubule-associated protein light chain 3; LCBF, local cerebral blood flow; MABP, mean arterial blood pressure; MCA, middle cerebral artery; NT, neurotensin; NTR1, neurotensin receptor 1; PIH, pharmacologically induced hypothermia; TTC, 2,3,5-triphenyltetrazolium chloride; TUNEL, terminal deoxynucleotidyl transferase biotin-dUPT nick end labeling; tPA, tissue plasminogen activator 0892-6638/12/0026-2799 © FASEB

Stroke is a leading cause of human death and disability in the United States and worldwide (1). Despite intensive basic and clinical investigations, thrombolytic therapy using tissue plasminogen activator (tPA) is so far the only clinically approved acute treatment for ischemic stroke. On the other hand, induction of hypothermia has proven effective in preclinical and clinical studies against brain damage induced by several insults, including ischemia (2– 4). Compelling evidence has shown that mild to moderate hypothermia (2–5°C reduction) is generally safe, protects brain tissues, and improves functional outcomes after cerebral ischemia (5, 6). Therapeutic hypothermia protects the brain in a comprehensive manner (7, 8). It reduces the energy demands of neuronal activity (9), attenuates free radical levels (10), and decreases extracellular levels of excitatory neurotransmitters, which helps to prevent excitotoxicity (9, 11). Hypothermia-induced neuroprotection is associated with suppressing apoptosis (12), as well as the inflammatory response in the ischemic brain (13). Moreover, it inhibits pathways related to blood-brain barrier (BBB) degeneration (8), decreases intracranial pressure (14), increases cerebral perfusion pressure (14), and reduces the risk of brain edema and hemorrhage (15, 16). In the clinic, therapeutic hypothermia is an approved therapy after cardiac arrest and in children with hypoxicischemic encephalopathy (17). Phase II and III clinical trials for stroke treatment have been carried out using physical cooling methods to induce hypothermia in patients (18). Although the mechanisms behind hypothermia-induced brain protection and functional benefits are still under investigation, clinical studies support the notion that temperature control is critical in the treatment of stroke. For example, a higher body temperature (noninfectious hyperthermia or pyrexia) of ⬃37– 40°C after ischemia is a common clinical symptom that worsens patient outcome (19). According to clinical data, post1 Correspondence: 101 Woodruff Cir., Woodruff Memorial Research Bldg., Ste. 620, Emory University School of Medicine, Atlanta, GA 30322, USA. E-mail: [email protected] doi: 10.1096/fj.11-201822

2799

stroke pyrexia may increase the rate of fatality by 43% (20). Consistently, temperature of ⬍36.5°C at admission is associated with reduced in-hospital mortality (21). Thus, therapeutic hypothermia is not only globally brain protective, but also prevents postischemia hyperthermia and pyrexia; either of these effects should improve the outcome of treatment for acute stroke (22). As a neuroprotective and preventive therapy, early hypothermia induction is an attractive and possible first-line treatment after a stroke attack. However, this can only be accomplished when effective and practical methods of regulated hypothermia are available. Unfortunately, current physical means of forced cooling do not satisfy the requisites for early treatment of acute stroke. Physical cooling in humans is slow and cumbersome, typically requiring 3– 6 h of surface cooling to reach the targeted mild-to-moderate hypothermia of 2–5°C reduction. Physical cooling almost always triggers shivering and vasoconstriction responses, which make reduction and accurate control of the patient’s temperature very challenging (23). More recently, regulated hypothermia induced by pharmacological reagents that target central thermoreceptors and reduce the “set-point” in the brain thermoregulatory center has been suggested as a more efficient and safer way of reducing brain and core body temperature (24). We refer to this approach as pharmacologically induced hypothermia (PIH) and demonstrate here its effectiveness in a mouse stroke model using a novel neurotensin (NT) analog, ABS-201. NT has been described as a neuroprotective hypothermic agent in gerbils (25), but it normally does not cross the BBB, as it is rapidly degraded by blood peptidases. The C-terminal hexapeptide of NT, ArgArg-Pro-Tyr-Ile-Leu (NT [8 –13]), has the essential structural elements for full biological activity (26). NT [8 –13] analogs that are biologically stable can penetrate the BBB (27). Specific analogs are being developed as potential antipsychotic (28) and analgesic (29) agents; these agents also induce hypothermia when administered at higher concentrations as agonists of the NT receptor subtype 1 (NTR1) located in the brain (30). We have generated novel compounds, such as ABS-201, that show high affinity for human NTR1, exhibit BBB permeability, and effectively induce regulated hypothermia in rats (28, 31) and monkeys (unpublished data). ABS-201 (Fig. 1) can induce regulated hypothermia by intravenous (i.v.), intraperitoneal (i.p.), or oral administration (28). The present study is the first effort to test a biologically stable NT [8 –13] analog for the treatment of stroke in a rodent model of focal cerebral ischemia. We hypothesized that ABS-201 can induce regulated hypothermia with reasonable cooling and rewarming rates, and, on the basis of previous observations with physical cooling, we further hypothesized that this centrally induced hypothermia therapy should be protective, even when initiated with a significant delay. The long-term functional benefit of this approach was also evaluated 21 d after stroke. 2800

Vol. 26

July 2012

Figure 1. NT [8 –13] analog ABS-201. The chemical structure of ABS-201.

MATERIALS AND METHODS NT analogs The NTR1 agonist ABS-201 was synthesized using procedures described previously (27, 28, 31). The chemical structure of ABS-201 is shown in Fig. 1. The affinity of ABS-201 for NTR-1 as a competitive agonist, its biological stability, and its ability to pass through the BBB when administered systemically were reported previously (27). Animals and focal cerebral ischemic stroke models Focal cerebral ischemic stroke was induced by right middle cerebral artery (MCA) occlusion in adult male C57BL/6 mice (8 –12 wk, 22–28 g), as described previously with minor modifications (32, 33). Animals were anesthetized with 1.5% isoflurane. Briefly, the right MCA was permanently ligated, accompanied by transient bilateral common carotid artery (CCA) ligations for 7 min. Rectal temperature in all groups was monitored and maintained at 37 ⫾ 0.5°C during surgery, using a heating pad controlled by a homeothermic blanket control unit (Havard Apparatus, Holliston, MA, USA). Animals in the normothermic group were injected with saline after stroke, and their body temperature was maintained at 36 –37°C in a humidity-controlled incubator (Thermocare, Incline Village, NV, USA) for up to 6 h after surgery. For the PIH experimental group, animals were subjected to ABS-201 injections, and no other methods were used to influence their body temperature. Body and brain temperature was monitored for ⱖ6 h after surgery. The animal protocol was approved by the Emory University Institutional Animal Care and Use Committee and met U.S. National Institutes of Health (NIH) guidelines. Food and water were available ad libitum for all animals. Drug administration and induction of hypothermia ABS-201 was dissolved in saline and injected intraperitoneally. To determine the dose-response relationship, ABS-201 was first tested at 1.5, 2.0, and 2.5 mg/kg in C57BL/6 mice without stroke surgery. For stroke treatment groups, the first bolus injection (2 mg/kg) was given immediately or at 30 or 60 min after CCA reperfusion, followed by additional injections at half of the initial dose (1 mg/kg). The interval between the following injections was ⬃1.5 h, with adjustments made in order to keep a constant mild hypothermia (33–

The FASEB Journal 䡠 www.fasebj.org

CHOI ET AL.

35°C). Rectal temperature was monitored using a rectal probe (Harvard Apparatus) for ⱖ6 h after ischemia, with measurements performed every 15 min for the first hour and every 30 min after. Brain temperature was monitored using a brain temperature probe (Physitemp, Clifton, NJ, USA) placed on the surface of the cerebral cortex through a 1-mm burr hole at the craniotomy site (34). Physical forced cooling was tested as a hypothermia control. The targeted body temperature was the same as in the PIH experiments (33°C). This was achieved by placing animals on ice during the first 15 min and in a ⱖ4°C chamber during the maintenance period. Compared to PIH, larger variations in body temperature (⫾1–2°C) occurred during physical cooling (see Fig. 2D). In experiments examining autophagic cell death, the autophagy inhibitor 3-methyladenine (3-MA; 15 mg/ml in 2 ␮l; Sigma, St. Louis, MO) was administered by intracerebroventricular (i.c.v.) injection (AP, ⫺0.23 mm; ML, ⫺1.0 mm; DV, ⫺2.2 to ⫺2.5 mm) through a Hamilton syringe (Hamilton Co., Reno, NV, USA) in a stereotactic apparatus immediately after CCA reperfusion. Saline (2 ␮l) was injected in the same i.c.v. space in control animals. The rectal temperature of both the 3-MA and saline i.c.v. groups was monitored in the same manner as animals in other groups. Similar to the normothermic control group, they were kept in a temperaturecontrolled incubator (Thermocare) after surgery, and their body temperature was strictly maintained at 36 –37°C. Brain tissue was immediately frozen in a dry ice box and embedded with optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) after sacrifice. Samples were stored at ⫺80°C until further analysis.

Local cerebral blood flow (LCBF) measurement The PeriScan PIM II laser-Doppler perfusion imager (Perimed AB, Cleveland, OH, USA) was used as described previously (32, 33) to measure LCBF in the penumbra region at 5 time points: before MCA occlusion, during MCA occlusion, and 16, 24, and 72 h after CCA reperfusion. Under anesthesia, the animal skull was fixed and exposed with a skin incision. The laser beam was centered at the middle of the right coronal suture (ML ⫹2.0 mm, AP ⫹1.0 mm), and blood perfusion images were collected in a 2.5 ⫻ 2.5 mm2 area over the penumbra region. This system measures the Doppler frequency shift generated by photons absorbed in moving blood cells (35). Tissue perfusion was calculated by the concentration of blood cells within the scattering volume times the average velocity, and the relative intensity of tissue perfusion was imaged by LDPIwin 2.6 software (Perimed) in a representative color scaling. To reduce measurement artifacts, 6 repeated images were obtained at the same scanned area with 10 ⫻ 10 measurement sites under high-resolution settings. Values are represented as a ratio to the baseline prestroke level for each animal, and the hypothermia group was compared to saline control group. Physiological parameter measurements To evaluate the effects of ABS-201 on physiological parameters, blood glucose level, blood pH, and mean arterial blood pressure (MABP) were monitored at different time points during of the experiments. Whole-blood samples were collected immediately and at 3 and 5 h after CCA reperfusion.

Figure 2. ABS-201 induced a dose-dependent hypothermic effect in mice. ABS-201 induces mild hypothermia in C57BL/6 mice. A) Dose-response curve of the ABS-201 effect. ABS-201 (i.p.) decreases core body (rectal) temperature in a dose-dependent manner in normal mice. *P ⬍ 0.05 vs. saline control; n ⫽ 4/group. B) PIH time course after ischemia. ABS201 induced hypothermia after cerebral ischemia. A bolus injection of ABS-201 (2 mg/kg, i.p.) was administered at the onset of CCA reperfusion (time 0), and it rapidly reduced body temperature to the range between 33°C and 35°C (dotted lines). Transient temperature drop to ⬍33°C was likely due to the concurrent effect of anesthesia. Consequent injections of ABS-201 at a half dose maintained the hypothermic effect for ⱖ6 h. *P ⬍ 0.05 vs. saline control; n ⱖ 7/group. C) Brain and body temperature measurements. Brain temperature reduction was similar to that of rectal temperature in both normothermia and hypothermia groups. For brain temperature measurements, the burr hole was not completely sealed. Lack of thermal insulation at the surface of the brain can explain difference between brain and rectal temperature. *P ⬍ 0.05 vs. normothermia brain temperature; n ⫽ 7/group. D) Comparison of temperature curves from saline control and physical cooling. Note that physical cooling caused larger fluctuations in the rectal temperature around 33°C. *P ⬍ 0.05 vs. saline control; n ⫽ 9/group. E) ABS-201 did not change blood glucose level during hypothermia after ischemia. P ⬎ 0.05 vs. saline (i.p.) control in each time point; n ⫽ 7/group. Values are expressed as means ⫾ se; all comparisons were analyzed using 2-way ANOVA followed by Bonferroni correction. PHARMACOLOGICAL HYPOTHERMIA FOR STROKE TREATMENT

2801

Blood glucose levels were measured using a blood glucose meter (Abbott, Alameda, CA, USA) and test strips (LifeScan, Milpitas, CA, USA). MABP was recorded using the Blood Pressure Analyzer-400 system (Digi-Med, Mobile, AL, USA) after left CCA catheterization with PE-10 polyethylene tubing (Becton Dickinson, Franklin Lakes, NJ, USA) under anesthesia before, immediately after, and at 1 and 2 h after ABS-201 injection. Animals were awake and freely moving between measurements, and the indwelling catheter was treated with heparin. Whole-blood samples were also collected to evaluate blood pH. Blood serum was separated by centrifugation at 17,000 g for 5 min at 4°C and was then applied on Hydrion pH test paper (Micro Essential Laboratory, Brooklyn, NY, USA) to measure the blood pH value. Quantification of infarct volume

Terminal deoxynucleotidyl transferase biotin-dUPT nickend labeling (TUNEL) A TUNEL assay kit (DeadEnd Fluorometric TUNEL system; Promega, Madison, WI, USA) was used to assess cell death by detecting fragmented DNA in 10-␮m-thick coronal fresh frozen sections. After fixation [10% buffered formalin for 10 min then ethanol:acetic acid (2:1) solution for 5 min] and permeabilization in 0.2% Triton-X 100 solution, brain sections were incubated in equilibration buffer for 10 min. Recombinant terminal deoxynucleotidyl transferase (rTdT) and nucleotide mixture were then added on the slide at 37°C for 60 min in the dark. Reactions were terminated by 2⫻ SSC solution for 15 min. Nuclei were counterstained with Hoechst 33342 (1:20,000; Molecular Probes, Eugene, OR, USA) for 5 min. Immunofluorescence staining Fresh frozen brain tissues were sliced into 10-␮m coronal sections using a cryostat vibratome (Leica CM 1950; Leica Microsystems, Buffalo Grove, IL, USA). Sections were dried on the slide warmer for 20 min, fixed with 10% buffered formalin, washed with ⫺20°C precooled ethanol:acetic acid (2:1) solution for 10 min, and finally permeabilized with 0.2% Triton-X 100 solution for 5 min. All slides were washed 3 times with PBS (5 min each) after each step. Then, tissue sections were blocked with 1% fish gelatin (Sigma) in PBS for 1 h at room temperature, and subsequently incubated with the primary antibodies microtubule-associated protein light chain 3 (LC3; 1:200; Novus, Littleton, CO, USA) and NeuN (1:300; Millipore, Billerica, MA, USA) overnight at 4°C. The following day, slides were washed 3 times with PBS for 5 min, then incubated with the secondary antibodies Cy3-conjugated donkey anti-rabbit (1:600, Jackson ImmunoResearch Laboratories, West Grove PA, USA) and Cy5-conjugated donkey anti-mouse (1:300; Jackson ImmunoResearch Laboratories) for 80 min at room temperature. After 3 washes with PBS, nuclei were stained with Hoechst 33342 (1:20000; Molecular Probes) for 5 min as a counterstain, then mounted with Vectashield fluorescent mounting medium (Vector LaboraVol. 26

July 2012

Quantification of positive cells Cell count was performed following the principles of designbased stereology. Systematic random sampling was employed to ensure accurate and nonredundant cell counting. Six brain sections (10 ␮m thick) per animal were collected at 100 ␮m distance between sections for nonoverlapping multistage random sampling. Six fields were chosen in each section in the penumbra region and viewed at ⫻40 for cell counting. ImageJ was used to analyze each picture. All analysis was performed in a blinded fashion. Western blot analysis

Infarct volume was assessed 24 h after ischemia by 2,3,5triphenyltetrazolium chloride (TTC) staining. Brains were removed and placed in a coronal brain matrix then sliced into 1-mm sections. Slices were incubated in 2% TTC (Sigma) solution at 37°C for 5 min, then stored in 10% buffered formalin for 24 h. Digital images of the caudal aspect of each slice were obtained by a flatbed scanner. Infarct, ipsilateral hemisphere, and contralateral hemisphere areas were measured using ImageJ software (NIH, Bethesda, MD, USA). Infarct volume was calculated using the indirect method (36).

2802

tory, Burlingame, CA), and coverslipped for microscopy and image analysis.

Expression of apoptotic and autophagic markers was assessed by Western blot analysis. Penumbra tissue samples were collected in the peri-infarct region and were homogenized to extract protein in lysis buffer [0.02 M Na4P2O7, 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA (pH 8.0), 1% Triton, 1 mM EGTA, 2 mM Na3VO4, and protease inhibitor cocktail (Sigma)]. The supernatant was collected after centrifugation at 17,000 g for 5 min at 4°C. Protein concentration was determined with a bicinchoninic acid (Pierce Biotechnology, Rockford, IL, USA) assay. Equivalent amounts of total protein were separated by molecular weight on SDS-PAGE gradient gel, then transferred to PVDF membranes. Transfer was followed by blocking with 10% skim milk in TBS with 0.1% Tween-20 solution (TBST) for 40 min. Membranes were incubated with primary antibodies overnight at 4°C as follows: rabbit anti-cleaved caspase-3 (CC3; 1:500, no. 9661; Cell Signaling, Danvers, MA, USA), rabbit antibody B-cell lymphoma 2 (bcl-2; 1:1000, 2876; Cell Signaling), rabbit anti-LC3 (1:1000, NB100-2331; Novus), rabbit anti-p62 (1:1000, 01822141; Wako, Richmond, VA, USA), mouse anti-␤-actin (1: 2000; Sigma). After 3 washes with TBST, membranes were incubated with AP-conjugated secondary antibodies (Promega) or HRP-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ, USA) for 2 h at room temperature. After a final wash with TBST, signals were detected with bromochloroindolyl phosphate/nitroblue tetrazolium (BCIP/NBT) solution (Sigma) or film. Signal intensity was measured by ImageJ and normalized to the ␤-actin signal intensity. Corner test behavior assay The corner test for whisker sensorimotor function analysis was performed at 3, 7, 14, and 21 d after ischemia, as described previously (37). Two cardboard plates (30⫻20⫻0.3 cm) were attached at a 30° angle from each other in a home cage. Each subject mouse was placed between the two plates and allowed to freely move to the corner. The numbers of right and left turns were counted. Ten trials/test were performed for each mouse. Statistics GraphPad Prism 4 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis and graphic presentation. Student’s 2-tailed t test was used for comparisons of 2 experimental groups, and a 1-way ANOVA followed by Bonferroni correction was used for multiple-group comparisons. A 2-way ANOVA followed by Bonferroni correction was used for repeated measurements. Significant differences between groups were identified by a value of P ⬍ 0.05. All data are presented as means ⫾ se.


CHOI ET AL.

RESULTS Pharmacological hypothermia induced by the NT analog ABS-201 Consistent with previous studies in rats (29), we confirmed that ABS-201 induced pharmacological hypothermia in adult C57BL/6 mice when administered intraperitoneally. We tested the efficacy and time course of 3 dosages of ABS-201, targeting mild to moderate hypothermia (2–5°C reduction in brain and core temperature). ABS-201 was administered by i.p. injection at 1.5, 2.0, or 2.5 mg/kg (Fig. 2A). Core body temperature dropped below 35°C within 15 min after injection in all three groups without detectable shivering. All animals were awake during the hypothermia treatment, but locomotor activity decreased as body temperature dropped. At the lowest dosage of 1.5 mg/kg, ABS-201 induced hypothermia was maintained for ⬍60 min, and temperature recovered back to ⬎36°C in 90 min with a rewarming rate of ⬃0.04°C/ min. At 2.0 mg/kg, mild PIH remained for ⬃60 min, with the lowest temperature reaching 33.1 ⫾ 0.7°C and a slower temperature recovery (Fig. 2A). At the highest dose of 2.5 mg/kg, ABS-201-induced hypothermia reached the lowest point of 31.8 ⫾ 0.5°C, and hypothermia was maintained for ⬎2 h after injection (Fig. 2A). The core body temperature of the control group was stable at 37.2 ⫾ 0.1°C after saline injection at room temperature. We then verified that ABS-201 effectively induced controlled hypothermia when administered in stroke animals and that the PIH effect could be prolonged with additional maintenance doses (Fig. 2B). Simultaneous measurements of both body and brain temperature demonstrated that changes in body temperature were highly correlated to changes in brain temperature (Fig. 2C). These observations indicate that pharmacological hypothermia can be quickly achieved in the brain and body (within 15–30 min of ABS-201 i.p. administration) and that the degree and maintenance of PIH, as well as the rewarming rate, can be controlled or manipulated by altering the dosage of the hypothermic compound. Physiological monitors showed that PIH had no significant effect on MABP, blood glucose, or blood pH (Table 1 and Fig. 2E). In stroke animals, we adopted the protocol of an initial bolus injection of 2 mg/kg ABS-201 followed by additional injections of 1 mg/kg of maintenance doses in

order to maintain the body temperature below 35°C for 6 h (Fig. 2B). This injection regimen was initiated either immediately or at 30 or 60 min after CCA reperfusion. The 6-h time point was chosen because this is the duration of physical hypothermia that shows noticeable neuroprotection against brain ischemic damage (38). This PIH protocol was then tested as a potential protective treatment for ischemic stroke in adult mice. Pharmacological hypothermia induced brain protection after ischemic stroke At 24 h after stroke, brain sections were analyzed for infarct formation. In TTC-staining assay, a 40.3% decrease in infarct volume was observed when PIH was initiated immediately after reperfusion, compared to saline normothermia stroke controls (Fig. 3A, B). In the PIH group, in which ABS-201 was injected with a 30-min delay, infarct volume was reduced by 33.4%. When administered with a 60-min delay, ABS-201 caused a 30.6% reduction in brain infarct formation (Fig. 3A, B). To verify that the neuroprotective effect of ABS-201 was due to its hypothermic action, rather than an off-target effect, we additionally tested a group of mice that received ABS-201 injections immediately after stroke and the same reinjection protocol. These mice, however, were kept in a temperature controlled incubator after ABS-201 injection in order to prevent induction of hypothermia. Temperature in this group was maintained at 36.2 ⫾ 0.1°C for 6 h, and infarct volume showed no reduction compared to stroke saline control animals (Fig. 3B). On the other hand, although physical cooling in this investigation showed a trend of reducing the infarct volume, the effect was not significant (Fig. 3). We observed that physical cooling in mice caused large and repetitive variations of the body temperature, accompanied by noticeable shivering of the animals (Fig. 3D). Pharmacological hypothermia inhibited apoptotic cell death in the ischemic brain To understand the mechanism of protective effect of ABS-201-induced hypothermia at the cellular level, we performed TUNEL staining in brain sections 6 –72 h after reperfusion. At all inspected time points, mice in the PIH group showed significantly fewer TUNELpositive cells in the penumbra region than that in the stroke saline control group (Fig. 4A, B, D). Counts of TUNEL-positive cells double stained with the neuro-

TABLE 1. Physiologic parameters after ABS-201 administration Time after injection Parameter

MABP (mmHg) Blood pH Core body temperature (°C)

Baseline

0 min

1h

2h

91.6 ⫾ 1.68 7.30 ⫾ 0.06 36.2 ⫾ 0.13

90.9 ⫾ 0.45 7.17 ⫾ 0.03 36.2 ⫾ 0.23

91.4 ⫾ 1.31 7.17 ⫾ 0.03 30.3 ⫾ 1.44

91.7 ⫾ 0.42 7.23 ⫾ 0.03 32.9 ⫾ 0.67

Values are expressed as means ⫾ se; n ⫽ 3-5 at each time point. MABP, mean arterial blood pressure. MABP and blood pH are not significantly changed after ABS-201 (i.p.) injection.

PHARMACOLOGICAL HYPOTHERMIA FOR STROKE TREATMENT

2803

Figure 3. ABS-201-induced hypothermia after ischemic stroke exhibited a neuroprotective effect. ABS-201-induced hypothermia initiated at 0, 30, or 60 min after CCA reperfusion reduced infarct volume. A) Representative brain sections of ischemic mice receiving different treatments. Infarct formation was examined using TTC staining at 24 h after ischemia. Mice receiving ABS-201 showed smaller infarct volumes compared to saline controls. B) Summarized TTC measurements of infarct volume. ABS-201 administered at 0, 30, or 60 min after CCA reperfusion significantly reduced infarct formation. As a control, a group of ABS-201-treated mice was kept in a temperature-controlled incubator to counteract its hypothermic effect, and body temperature was maintained at 36 –37°C during and after ischemia. Mice in this group developed brain infarct volumes similar to saline controls. Autophagy inhibitor 3-MA (2 ␮l, i.c.v., 15 mg/ml applied immediately after CCA reperfusion) also reduced infarct volume compared with the saline i.c.v. injection group. All animals subjected to 3-MA and saline i.c.v. injection maintained their body temperature at 36 –37°C during and after ischemia. Physical surface cooling was tested (targeting 33°C for 6 h, initiated on CCA reperfusion) and showed a comparable neuroprotective effect of reducing infarct volume 24 h after ischemia. In panel A, brain section alignment of the saline group was adjusted using Adobe Photoshop CS4 (Adobe Systems, San Jose, CA, USA). *P ⬍ 0.05 vs. saline (i.p.) control; #P ⬍ 0.05 vs. saline (i.c.v.) control; n ⫽ 9 –15/group.

nal marker NeuN indicated that the PIH therapy reduced neuronal cell death (Fig. 4C, E). Expression of the antiapoptotic gene bcl-2 in the postischemic brain of PIH animals was significantly higher than its expression in the stroke control animals (Fig. 5C). In addition, activity of the key apoptotic enzyme caspase-3 was suppressed by PIH (Fig. 5D).

Pharmacological hypothermia suppressed autophagic cell death in the ischemic brain Since recent evidence indicates that autophagy may contribute to ischemia-induced neuronal injury after ischemic stroke (39), we tested the possibility that our PIH therapy might suppress autophagic activity in the

Figure 4. ABS-201 reduced ischemiainduced cell death. TUNEL staining was performed to detect DNA damage and cell death in the ischemic core and penumbra regions of brain sections. Neuronal cells were identified by NeuN staining, whereas the nuclei of all cells were visualized with Hoechst staining. A, B) TUNEL-positive cells (green) were reduced in the ABS-201-treated hypothermia group (B) compared with the control group (A). C) TUNEL (green) and NeuN (red) double-positive cells represent neuronal cell death in the penumbra. Neuronal cell death was reduced in the ABS-201-treated hypothermia group. D, E) Summarized data from experiments in panels A–C. Total cell death (TUNEL/Hoechst positive; D) and neuronal cell death (TUNEL/NeuN positive; E) were attenuated at various time points by ABS-201-induced hypothermia. *P ⬍ 0.05; n ⫽ 4 –5/group. Scale bars ⫽ 100 ␮m (A, B); 20 ␮m (C, D). 2804

Vol. 26

July 2012


CHOI ET AL.

Figure 5. ABS-201-reduced ischemia-induced autophagic damage. Expression of proteins associated with autophagy and apoptosis were measured by Western blot analysis in the penumbra region at 24 h after stroke. A) ABS-201 hypothermia and 3-MA treatment groups showed decreased LC3 II expression. B) Hypothermia increased the level of p62 compared to the saline normothermia group, suggesting suppression of excessive postischemic autophagy flux. C, D) Hypothermia and 3-MA treatment groups showed increased levels of the antiapoptotic protein bcl-2 (C) and decreased levels of CC3 (D). *P ⬍ 0.05; n ⫽ 4 – 6/group, except sham group (n⫽3).

postischemic brain. Supporting the idea that excessive autophagy is an injurious mechanism in the ischemic brain, the autophagy inhibitor 3-MA (15 mg/ml, 2 ␮l, i.c.v. injection, immediately after CCA reperfusion) significantly reduced brain infarct volume (Fig. 3). Autophagic activity was examined by the formation of LC3 II in the penumbra region using Western blot analysis (40). Increased LC3 II expression was observed 24 h after ischemia, and both ABS-201 and 3-MA treatments eliminated the LC3 II increase (Fig. 5A). In addition, degradation levels of sequestosome 1/p62, a shuttle protein interacting directly with LC3 (41), were decreased by PIH in the ischemic brain, indicating an inhibitory effect of PIH on autophagy (Fig. 5B). In

immunofluorescent staining, LC3-labeled autophagosome formation was decreased by ABS-201 or 3-MA treatment (Fig. 6). TUNEL/LC3/NeuN triple-labeled cells confirmed that autophagy contributed to neuronal cell death in the postischemic brain (Fig. 6A). Total numbers of TUNEL-positive cells and TUNEL/LC3/ NeuN triple-positive cells were attenuated by ABS-201 or 3-MA treatments (Fig. 6E–G). TUNEL-positive nuclei colocalized with autophagic markers had a different morphology than those that did not colocalize. Some TUNEL-positive cells resembled typical apoptotic cells, with chromatin condensation and nuclear fragmentation, while others showed little chromatin clumping within an intact nucleus (Fig.

Figure 6. Morphology of LC3- and TUNEL-positive cells. Morphological features of apoptotic and authophagic cell death in the penumbra 24 h after ischemia were examined. A) Many LC3-positive (red) vacuoles overlap with TUNEL-positive (green) and NeuN-positive (blue) cells, suggesting an autophagic component in the cell death (arrows). B) Confocal image showing LC3- and TUNEL-positive cells with a 3-dimensional view. C) Demonstration of two different morphologies of TUNEL-positive nuclei. Some cells have no karyorrhexis and little chromatin clumping with light green fluorescence (arrow), and others exhibit obvious chromatin condensation with karyorrhexis with a stronger green fluorescent signal (arrowhead). D) TUNEL/LC3/NeuN triple-stained cells are reduced in the penumbra in both the ABS-201 and 3-MA treatment groups. E–G) Summarized cell counts in TUNEL-positive (E), TUNEL/NeuN-positive (F), and TUNEL/LC3/NeuN-positive cells (G). TUNEL/LC3/NeuN-positive cells were decreased in both the ABS-201 (2 mg/kg, i.p.) hypothermia- and 3-MA (15 mg/ml, 2 ␮l, i.c.v. injection in each animal)-treated groups. Scale bars ⫽ 20 ␮m (A, D); 10 ␮m (B, C). *P ⬍ 0.05, **P ⬍ 0.01; n ⫽ 4/group. PHARMACOLOGICAL HYPOTHERMIA FOR STROKE TREATMENT

2805

6C). The latter were labeled with a less intense and more diffuse fluorescent signal, implying diffuse fragments of DNA nick-end labeling. Only the less intense and more diffuse fluorescent TUNEL-positive cells (named type I TUNEL-positive cells in our investigations; ref. 42) costained for LC3-positive vacuoles in the cytoplasm (Fig. 6A, B). Pharmacological hypothermia did not alter local cerebral blood flow in the ischemic brain Since physical cooling may cause a decrease in cerebral blood flow (43), it was necessary to confirm possible alteration of the LCBF in response to the hypothermic compound ABS-201. LCBF at the ischemic core and penumbra was measured using the laser-Doppler scanning method before and during ischemia, as well as at several time points after reperfusion. The laser-Doppler scanner surveys an area of 2.5 ⫻ 2.5 mm2 centered at AP ⫹ 1.0 mm and ML 2.0 mm from the bregma. Local flow in the tissue perfused by the right MCA was dramatically decreased by 80 –90% in all animals during MCA occlusion and there was no further decrease of LCBF in the hypothermia group during ischemia or after reperfusion (Fig. 7). Pharmacological hypothermia promoted functional recovery after ischemic stroke The corner test is a sensitive and specific test for sensorimotor function involving the whisker barrel cortex and its thalamocortical connections (37). In normal rats or mice, whiskers on both sides are used for exploratory activities. Thus, normal animals make equal left and right turns in the corner test. After ischemic damage to the right side of the barrel cortex, subject animals have impaired left-side whisker sensation due to the right-side stroke and rely on the intact right-side whisker sensations for exploration; this results in a preferential turning to the right side in the corner test (37). In mice that received ABS-201 treatment, whisker activity was significantly improved 21 d after stroke, with animals showing less preference for turning to the right side of the corner as compared to animals in the stroke saline group (Fig. 8).

DISCUSSION The present investigation evaluated the potential of the NTR1 agonist ABS-201 as a novel pharmacological agent for a PIH therapy after acute ischemic stroke. We show that ABS-201 treatment effectively reduced body and brain temperature. When administrated either immediately or up to 60 min after stroke attack, ABS201-induced PIH was clearly effective in reducing infarct formation and brain cell death. The PIH therapy is also effective in promoting long-term functional recovery in poststroke animals. The treatment takes the advantage that ABS-201 can penetrate the BBB and 2806

Vol. 26

July 2012

Figure 7. LCBF restoration in the penumbra is unaffected by PIH. LCBF was measured using the PeriScans laser image scanner before, during ischemia (MCA/CCA occlusion), and 16, 24, and 72 h after CCA reperfusion. A) Diagram shows the area (2.5⫻2.5 mm2 centered at the middle of the right coronal suture ⫺AP ⫹1.0 mm, ML ⫹2.0 mm) covering the penumbra in our stroke model, identified using the stereotactic procedure. Same area (arrow) was scanned at different time points. Color scale shows the relative value from the lowest perfusion (blue, 0) to the highest perfusion (red, 10) of LCBF. Scanning images show LCBF in the same location before, during, and at different times after ischemia in 2 experimental groups. B) Line graph shows values quantified relative to prestroke baseline measurements. LCBF in the ABS-201 group is not significantly different from the saline control group. Values are expressed as means ⫾ se; n ⫽ 7–25/group at each time point.

centrally induces controlled hypothermia by downregulating the temperature set-point in the brain after systemic administration (31). The onset of hypothermia after ABS-201 administration was fast (within 15 min), and animals subjected to ABS-201 did not exhibit shivering or consequent rebound hyperthermia, which can deteriorate stroke outcome. The degree and duration of hypothermia induced by ABS-201 can be controlled by the dosage and injection protocol of the compound. Animals treated with ABS-201 showed decreased locomotor activity during the hypothermic period, as described previously (28), but were alert during hypothermia and recovered normal locomotion on rewarming. NT derivatives may also cause antinociceptive and antipsychotic effects (44, 45), but these are nonlethal and readily reversible. It is expected that, if encountered, these side effects should be tolerable during acute treatment of a few hours. Hypothermia reduces cerebral blood flow in the


CHOI ET AL.

absence of ischemia (43). This raised the concern that PIH would have a negative effect on already reduced LCBF during and after focal ischemia. Our data showed that LCBF in the ischemic region of ABS-201-treated animals was not significantly different from normothermic animals. This is consistent with previous reports that therapeutic hypothermia did not further decrease cerebral blood flow either during or after cerebral ischemia (46). On the other hand, side effects can occur as a result of severe hypothermia (⬍30°C). For example, hypothermia can increase or decrease blood glucose levels depending on the degree of temperature reduction and tends to increase blood pH values (43, 47), although it may not affect MABP (43, 48). At the dosages used to induce therapeutic hypothermia, ABS201 did not significantly change MABP, blood glucose levels, or blood pH. These observations are important in light of a recent report that administration of adenosine 5=-monophosphate (AMP), which was tested as a hypothermia reagent in rats, causes severe hyperglycemia, hypotension, and hypoperfusion that may be responsible for exaggerated brain damage after stroke (47). The dissimilar effects of AMP and ABS-201 on physiological parameters were likely due to their completely different receptor targets and different mechanisms of action. Recent studies suggest that excessive autophagy flux can cause cell death in the ischemic penumbra (49, 50), and autophagy inhibition by 3-MA can alleviate cell death and injury (50). The effect of hypothermia on autophagic cell death, however, has not been investigated before. In the present study, we hypothesized that PIH therapy could be neuroprotective partly due to

suppression of excessive postischemia autophagic flux. In agreement with this hypothesis, we presented data that LC3 II-positive cells increased in the normothermia group, and these cells costained with TUNEL. As a general concession for measuring autophagy, more than one marker and signaling pathway was assessed (51), and we showed that autophagic activity in the penumbra was attenuated by ABS-201 treatment. The 3-MA results support the idea that inhibition of autophagy is neuroprotective after focal ischemia. It is well known that TUNEL assay can detect both apoptotic and nonapoptotic cell death; our investigations have suggested that nuclear morphology should be considered when using this assay (42). The nuclear morphology of LC3-positive cells shows no nuclear fragmentation or pyknotic nuclei and has little chromatin clumping, which is similar to previous reports (52). In the present study, two distinct morphologies of TUNEL-positive cells matching our classification of type I and type II TUNEL-positive cells were observed (42). LC3-positive cells with more diffuse TUNEL staining resemble the type I TUNEL-positive cells (42), which have no karyorrehxis, no pyknosis of the nuclei, and no heavy chromatin condensation. The type II TUNEL-positive cells, which have nuclear fragmentation, pyknotic nuclei, and heavy chromatin condensation, were brightly stained in the TUNEL assay and did not overlap with LC3 staining (42). Our observations also agree with previous reports showing that heavy chromatin condensation does not occur in autophagic cells (53), even though DNA damage can be detected (54, 55). Previous to this study, several NT-based compounds were evaluated as possible hypothermic agents. The NT analogs NT69L (56) and NT77 (24, 57) induced hypothermia when injected intravenously and showed a neuroprotective effect after asphyxia and cardiac arrest. JMV449, a pseudopeptide analog of NT [8 –13], induced hypothermia after i.c.v. administration and decreased infarct volume after permanent cerebral ischemia in mice (58). However, each of these compounds has important shortcomings. Hypothermic tolerance develops after a single dose of NT69L (59, 60), in contrast to ABS-201, which maintained hypothermic induction after repeated i.p. administration (28). Hypothermia induced by NT77 could not be counteracted by significant rewarming measures, and this drawback increased mortality (57). JMV449 is unable to penetrate the BBB and is therefore not clinically practical. We noticed that none of these dilemmas was observed with ABS-201 in our investigation. Currently, tPA is the only FDA-approved treatment for acute stroke. However, tPA treatment requires diagnosis and exclusion of hemorrhagic stroke, which can only be accomplished after arrival of patients at a hospital equipped with a specialized stroke center. The majority of patients either miss the therapeutic window (ⱕ4.5 h after the onset of stroke) or are excluded due to the hemorrhagic risk. Therefore, a treatment effective for both ischemic and hemorrhagic attacks would


2807

Figure 8. ABS-201 improved functional recovery after ischemic stroke. Corner test was used to evaluate the functional integrity of the whisker-barrel pathway up to 21 d after stroke. Normal animals use whiskers on both sides for exploratory activity, illustrated by equal right and left turns distributed around the line of 0.5. Stroke damage to one side of the sensorimotor cortex results in a noticeable bias in turning to one direction, resulting in up-shifts from the middle line in the figure. Stroke animals that received ABS-201 showed less bias in turns at all time points tested; calculated values scattered more around the middle line than stroke only controls. At 21 d after stroke, mice in the hypothermia group showed significantly less preference in turning as compared to control group. *P ⬍ 0.05; n ⫽ 3–5/group.

have an enormous advantage for early intervention. Previous evidence showed that early mild hypothermia induced by physical means is beneficial not only for ischemic stroke but also for hemorrhagic stroke (61– 63), Our most recent study of hemorrhagic stroke in mice consistently demonstrates protective effects of ABS-201 against hemorrhage-induced brain damage (64). Thus, ABS-201 could be an emergency onsite treatment for acute stroke without requirement of immediate identification of ischemic and hemorrhagic pathogenesis. The early administration of PIH therapy should have at least 3 clinical benefits: first to protect brain cells of different phenotypes, second, to prevent postischemic hyperthermia/pyrexia, and finally, to prolong the therapeutic window for tPA treatment. The last potential provides a feasible combination therapy but remains to be specifically investigated. Although up to a 60-min delay of PIH therapy was tested in the present investigation, which is expected to be clinically relevant as an on-site treatment, a previous report on physical cooling in stroke rats showed that hypothermia was still significantly neuroprotective when initiated 4 h after reperfusion (65). It is possible that PIH therapy could have a similar therapeutic window for ischemic stroke. This possibility should be tested in different stroke models with different duration of PIH therapy. It is also important to recognize that the metabolic rate and pathological evolution of ischemic damage in rodents are faster than that in humans; thus, the therapeutic window for humans is expected to be longer than the 1-h delay tested in this investigation. An accurate estimation of the PIH therapeutic window should be better determined using a nonhuman primate model of stroke and verified in human patients. Since shivering and vasoconstriction are associated with physical cooling methods, physical cooling has to be delayed until general anesthesia is available to the patient (23). Recent methods using i.v. heat exchange or infusion (66) act much faster than surface cooling, but shivering remains a major dilemma. In addition, these methods require highly specialized cooling technologies and specially trained personnel that are not available to most patients treated for acute stroke. Because of the great need for stroke therapy, alternative methods of reducing temperature in humans have been evaluated. For example, antipyretic drugs, such as paracetamol, can induce hypothermia (59); however, these drugs are ineffective in inducing hypothermia, promoting only a ⬃1°C decrease in core body temperature, even in combination with forced-air surface cooling. This minor effect apparently is not sufficient for an effective hypothermic therapy (67). In a recent review article, 5 pharmacological and 3 physical temperature reduction clinical trials involving a total of 423 patients were summarized (68). The pharmacological hypothermia trials evaluated 5–24 h poststroke administrations of the analgesic or antipyretic drugs aceteminophen (paracetamol), metamizole, and ibuprofen. These treatments did not show any improvement in 2808

Vol. 26

July 2012

1– 6-mo outcomes (death and dependency) of the patients. We took a close examination on the report and noticed that the mean body temperature of treated patients in these trials was reduced only by 0.2°C compared to control groups. The pharmacological methods used in these trials cannot be considered true hypothermia therapy, although they may have some value in antagonizing poststroke pyrexia. They also differ fundamentally in the mechanism of action from the NT receptor-mediated hypothermia tested in our investigation. The physical hypothermia trials that employed surface cooling methods were slow and largely ineffective. These clinical trials, therefore, are not representative of the current advances in hypothermia therapy. Among many experimental stroke therapies, mild to moderate hypothermia remains a prominent potential therapy for stroke (69). On the basis of numerous preclinical and clinical studies using physical cooling methods and on the basis of effectiveness and advantages of PIH treatment shown in this investigation, we expect that the novel PIH therapy has an encouraging potential for the treatment of acute stroke in clinical settings. Importantly, reduction of brain temperature should provide a global brain protection of both neurons and non-neuronal cells. This concept of brain protection as opposed to neuroprotection is of clinical significance yet remains to be specifically evaluated. The possibility that PIH compounds may be beneficial for traumatic disorders, such as traumatic brain injury and spinal cord injury, also merits further investigations. In addition to the present investigation in which ABS-201 was administered by i.p. injection, various routes of ABS-201 administration are feasible, including i.v., intranasal, and possibly oral administration. Each of these may have clinical significance and should be specifically tested in future investigations. This work was supported by U.S. National Institutes of Health (NIH) grants NS057255, NS058710, NS062097, R41NS073378 (S.P.Y., L.W.), GM079044 (S.P.Y., T.A.D.), and MH65099 (T.A.D.) and by a Yerkes National Primate Center/ NIH P51 pilot grant. This work was also supported by NIH grant C06 RR015455 from the Extramural Research Facilities Program of the National Center for Research Resources.

REFERENCES 1.

Roger, V. L., Go, A. S., Lloyd-Jones, D. M., Benjamin, E. J., Berry, J. D., Borden, W. B., Bravata, D. M., Dai, S., Ford, E. S., Fox, C. S., Fullerton, H. J., Gillespie, C., Hailpern, S. M., Heit, J. A., Howard, V. J., Kissela, B. M., Kittner, S. J., Lackland, D. T., Lichtman, J. H., Lisabeth, L. D., Makuc, D. M., Marcus, G. M., Marelli, A., Matchar, D. B., Moy, C. S., Mozaffarian, D., Mussolino, M. E., Nichol, G., Paynter, N. P., Soliman, E. Z., Sorlie, P. D., Sotoodehnia, N., Turan, T. N., Virani, S. S., Wong, N. D., Woo, D., and Turner, M. B. (2012) Heart disease and stroke statistics–2012 update: a report from the American Heart Association. Circulation 125, e2–e220 2. Froehler, M. T., and Ovbiagele, B. (2010) Therapeutic hypothermia for acute ischemic stroke. Expert Rev. Cardiovasc. Ther. 8, 593–603


CHOI ET AL.

3. 4. 5.

6. 7. 8. 9. 10.

11.

12.

13. 14.

15. 16.

17.

18. 19. 20. 21. 22. 23.

24. 25.

Miyazawa, T., Tamura, A., Fukui, S., and Hossmann, K. A. (2003) Effect of mild hypothermia on focal cerebral ischemia. Review of experimental studies. Neurol. Res. 25, 457–464 Lampe, J. W., and Becker, L. B. (2010) State-of-the-art in therapeutic hypothermia. Annu. Rev. Med. 62, 79 –93 Van der Worp, H. B., Macleod, M. R., and Kollmar, R. (2010) Therapeutic hypothermia for acute ischemic stroke: ready to start large randomized trials? J. Cereb. Blood Flow Metab. 30, 1079 –1093 Yenari, M. A., and Hemmen, T. M. (2010) Therapeutic hypothermia for brain ischemia: where have we come and where do we go? Stroke 41, S72–S74 Gonzalez-Ibarra, F. P., Varon, J., and Lopez-Meza, E. G. (2011) Therapeutic hypothermia: critical review of the molecular mechanisms of action. Front. Neurol. 2, 4 Hammer, M. D., and Krieger, D. W. (2003) Hypothermia for acute ischemic stroke: not just another neuroprotectant. Neurologist 9, 280 –289 Schwab, S., Spranger, M., Aschoff, A., Steiner, T., and Hacke, W. (1997) Brain temperature monitoring and modulation in patients with severe MCA infarction. Neurology 48, 762–767 Globus, M. Y., Alonso, O., Dietrich, W. D., Busto, R., and Ginsberg, M. D. (1995) Glutamate release and free radical production following brain injury: effects of posttraumatic hypothermia. J. Neurochem. 65, 1704 –1711 Illievich, U. M., Zornow, M. H., Choi, K. T., Scheller, M. S., and Strnat, M. A. (1994) Effects of hypothermic metabolic suppression on hippocampal glutamate concentrations after transient global cerebral ischemia. Anesth. Analg. 78, 905–911 Maier, C. M., Ahern, K., Cheng, M. L., Lee, J. E., Yenari, M. A., and Steinberg, G. K. (1998) Optimal depth and duration of mild hypothermia in a focal model of transient cerebral ischemia: effects on neurologic outcome, infarct size, apoptosis, and inflammation. Stroke 29, 2171–2180 Yanagawa, Y., Kawakami, M., and Okada, Y. (2002) Moderate hypothermia alters interleukin-6 and interleukin-1alpha reactions in ischemic brain in mice. Resuscitation 53, 93–99 Schwab, S., Schwarz, S., Spranger, M., Keller, E., Bertram, M., and Hacke, W. (1998) Moderate hypothermia in the treatment of patients with severe middle cerebral artery infarction. Stroke 29, 2461–2466 Linares, G., and Mayer, S. A. (2009) Hypothermia for the treatment of ischemic and hemorrhagic stroke. Crit. Care Med. 37, S243–S249 Thome, C., Schubert, G. A., and Schilling, L. (2005) Hypothermia as a neuroprotective strategy in subarachnoid hemorrhage: a pathophysiological review focusing on the acute phase. Neurol. Res. 27, 229 –237 Nagel, S., Papadakis, M., Hoyte, L., and Buchan, A. M. (2008) Therapeutic hypothermia in experimental models of focal and global cerebral ischemia and intracerebral hemorrhage. Expert Rev. Neurother. 8, 1255–1268 Macleod, M. R., Petersson, J., Norrving, B., Hacke, W., Dirnagl, U., Wagner, M., and Schwab, S. (2010) Hypothermia for stroke: call to action 2010. Int. J. Stroke 5, 489 –492 Ginsberg, M. D., and Busto, R. (1998) Combating hyperthermia in acute stroke: a significant clinical concern. Stroke 29, 529 –534 Hajat, C., Hajat, S., and Sharma, P. (2000) Effects of poststroke pyrexia on stroke outcome: a meta-analysis of studies in patients. Stroke 31, 410 –414 Wang, Y., Lim, L. L., Levi, C., Heller, R. F., and Fisher, J. (2000) Influence of admission body temperature on stroke mortality. Stroke 31, 404 –409 Tyler-McMahon, B. M., Boules, M., and Richelson, E. (2000) Neurotensin: peptide for the next millennium. Regul. Pept. 93, 125–136 Schwab, S., Schwarz, S., Aschoff, A., Keller, E., and Hacke, W. (1998) Moderate hypothermia and brain temperature in patients with severe middle cerebral artery infarction. Acta Neurochir. Suppl. 71, 131–134 Katz, L. M., Young, A. S., Frank, J. E., Wang, Y., and Park, K. (2004) Regulated hypothermia reduces brain oxidative stress after hypoxic-ischemia. Brain Res. 1017, 85–91 Babcock, A. M., Baker, D. A., Hallock, N. L., Lovec, R., Lynch, W. C., and Peccia, J. C. (1993) Neurotensin-induced hypothermia prevents hippocampal neuronal damage and increased


26. 27.

28.

29.

30.

31.

32.

33.

34.

35. 36.

37.

38.

39. 40. 41.

42. 43. 44.

locomotor activity in ischemic gerbils. Brain Res. Bull. 32, 373–378 Carraway, R., and Leeman, S. E. (1975) The synthesis of neurotensin. J. Biol. Chem. 250, 1912–1918 Kokko, K. P., Hadden, M. K., Price, K. L., Orwig, K. S., See, R. E., and Dix, T. A. (2005) In vivo behavioral effects of stable, receptor-selective neurotensin[8 –13] analogues that cross the blood-brain barrier. Neuropharmacology 48, 417–425 Hadden, M. K., Orwig, K. S., Kokko, K. P., Mazella, J., and Dix, T. A. (2005) Design, synthesis, and evaluation of the antipsychotic potential of orally bioavailable neurotensin (8 –13) analogues containing non-natural arginine and lysine residues. Neuropharmacology 49, 1149 –1159 Hughes, F. M., Jr., Shaner, B. E., May, L. A., Zotian, L., Brower, J. O., Woods, R. J., Cash, M., Morrow, D., Massa, F., Mazella, J., and Dix, T. A. (2010) Identification and functional characterization of a stable, centrally active derivative of the neurotensin (8 –13) fragment as a potential first-in-class analgesic. J. Med. Chem. 53, 4623–4632 Dubuc, I., Sarret, P., Labbe-Jullie, C., Botto, J. M., Honore, E., Bourdel, E., Martinez, J., Costentin, J., Vincent, J. P., Kitabgi, P., and Mazella, J. (1999) Identification of the receptor subtype involved in the analgesic effect of neurotensin. J. Neurosci. 19, 503–510 Orwig, K. S., Lassetter, M. R., Hadden, M. K., and Dix, T. A. (2009) Comparison of N-terminal modifications on neurotensin(8 –13) analogues correlates peptide stability but not binding affinity with in vivo efficacy. J. Med. Chem. 52, 1803–1813 Li, Y., Lu, Z., Keogh, C. L., Yu, S. P., and Wei, L. (2007) Erythropoietin-induced neurovascular protection, angiogenesis, and cerebral blood flow restoration after focal ischemia in mice. J. Cereb. Blood Flow Metab. 27, 1043–1054 Ogle, M. E., Gu, X., Espinera, A. R., and Wei, L. (2012) Inhibition of prolyl hydroxylases by dimethyloxaloylglycine after stroke reduces ischemic brain injury and requires hypoxia inducible factor-1alpha. Neurobiol. Dis. 45, 733–742 Miyazawa, T., and Hossmann, K. A. (1992) Methodological requirements for accurate measurements of brain and body temperature during global forebrain ischemia of rat. J. Cereb. Blood Flow Metab. 12, 817–822 Humeau, A., Steenbergen, W., Nilsson, H., and Stromberg, T. (2007) laser-Doppler perfusion monitoring and imaging: novel approaches. Med. Biol. Eng. Comput. 45, 421–435 Swanson, R. A., Morton, M. T., Tsao-Wu, G., Savalos, R. A., Davidson, C., and Sharp, F. R. (1990) A semiautomated method for measuring brain infarct volume. J. Cereb. Blood Flow Metab. 10, 290 –293 Zhang, L., Schallert, T., Zhang, Z. G., Jiang, Q., Arniego, P., Li, Q., Lu, M., and Chopp, M. (2002) A test for detecting long-term sensorimotor dysfunction in the mouse after focal cerebral ischemia. J. Neurosci. Methods 117, 207–214 van der Worp, H. B., Sena, E. S., Donnan, G. A., Howells, D. W., and Macleod, M. R. (2007) Hypothermia in animal models of acute ischaemic stroke: a systematic review and meta-analysis. Brain 130, 3063–3074 Rami, A., Langhagen, A., and Steiger, S. (2008) Focal cerebral ischemia induces upregulation of Beclin 1 and autophagy-like cell death. Neurobiol. Dis. 29, 132–141 Mizushima, N., and Yoshimori, T. (2007) How to interpret LC3 immunoblotting. Autophagy 3, 542–545 Pankiv, S., Clausen, T. H., Lamark, T., Brech, A., Bruun, J. A., Outzen, H., Overvatn, A., Bjorkoy, G., and Johansen, T. (2007) p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J. Biol. Chem. 282, 24131–24145 Wei, L., Ying, D. J., Cui, L., Langsdorf, J., and Yu, S. P. (2004) Necrosis, apoptosis and hybrid death in the cortex and thalamus after barrel cortex ischemia in rats. Brain Res. 1022, 54 –61 Erecinska, M., Thoresen, M., and Silver, I. A. (2003) Effects of hypothermia on energy metabolism in mammalian central nervous system. J. Cereb. Blood Flow Metab. 23, 513–530 Nemeroff, C. B., Osbahr, A. J., 3rd, Manberg, P. J., Ervin, G. N., and Prange, A. J., Jr. (1979) Alterations in nociception and body temperature after intracisternal administration of neurotensin, beta-endorphin, other endogenous peptides, and morphine. Proc. Natl. Acad. Sci. U. S. A. 76, 5368 –5371

2809

45. 46.

47.

48.

49.

50. 51.

52.

53. 54. 55. 56. 57. 58.

2810

Nemeroff, C. B. (1980) Neurotensin: perchance an endogenous neuroleptic? Biol. Psychiatry 15, 283–302 Zhao, H., Steinberg, G. K., and Sapolsky, R. M. (2007) General versus specific actions of mild-moderate hypothermia in attenuating cerebral ischemic damage. J. Cereb. Blood Flow Metab. 27, 1879 –1894 Zhang, F., Wang, S., Luo, Y., Ji, X., Nemoto, E. M., and Chen, J. (2009) When hypothermia meets hypotension and hyperglycemia: the diverse effects of adenosine 5=-monophosphate on cerebral ischemia in rats. J. Cereb. Blood Flow Metab. 29, 1022– 1034 Battin, M. R., Thoresen, M., Robinson, E., Polin, R. A., Edwards, A. D., and Gunn, A. J. (2009) Does head cooling with mild systemic hypothermia affect requirement for blood pressure support? Pediatrics 123, 1031–1036 Tian, F., Deguchi, K., Yamashita, T., Ohta, Y., Morimoto, N., Shang, J., Zhang, X., Liu, N., Ikeda, Y., Matsuura, T., and Abe, K. (2010) In vivo imaging of autophagy in a mouse stroke model. Autophagy 6, 1107–1114 Puyal, J., Vaslin, A., Mottier, V., and Clarke, P. G. (2009) Postischemic treatment of neonatal cerebral ischemia should target autophagy. Ann. Neurol. 66, 378 –389 Martinet, W., De Meyer, G. R., Andries, L., Herman, A. G., and Kockx, M. M. (2006) Detection of autophagy in tissue by standard immunohistochemistry: possibilities and limitations. Autophagy 2, 55–57 Ginet, V., Puyal, J., Clarke, P. G., and Truttmann, A. C. (2009) Enhancement of autophagic flux after neonatal cerebral hypoxiaischemia and its region-specific relationship to apoptotic mechanisms. Am. J. Pathol. 175, 1962–1974 Kroemer, G., and Levine, B. (2008) Autophagic cell death: the story of a misnomer. Nat. Rev. Mol. Cell. Biol. 9, 1004 –1010 Shoji, J. Y., Kikuma, T., Arioka, M., and Kitamoto, K. (2010) Macroautophagy-mediated degradation of whole nuclei in the filamentous fungus Aspergillus oryzae. PLoS One 5, e15650 Dawaliby, R., and Mayer, A. (2010) Microautophagy of the nucleus coincides with a vacuolar diffusion barrier at nuclearvacuolar junctions. Mol. Biol. Cell 21, 4173–4183 Katz, L. M., Wang, Y., McMahon, B., and Richelson, E. (2001) Neurotensin analog NT69L induces rapid and prolonged hypothermia after hypoxic ischemia. Acad. Emerg. Med. 8, 1115–1121 Katz, L. M., Young, A., Frank, J. E., Wang, Y., and Park, K. (2004) Neurotensin-induced hypothermia improves neurologic outcome after hypoxic-ischemia. Crit. Care Med. 32, 806 –810 Torup, L., Borsdal, J., and Sager, T. (2003) Neuroprotective effect of the neurotensin analogue JMV-449 in a mouse model

Vol. 26

July 2012

59.

60.

61. 62.

63.

64.

65.

66. 67. 68. 69.

of permanent middle cerebral ischaemia. Neurosci. Lett. 351, 173–176 Tyler-McMahon, B. M., Stewart, J. A., Farinas, F., McCormick, D. J., and Richelson, E. (2000) Highly potent neurotensin analog that causes hypothermia and antinociception. Eur. J. Pharmacol. 390, 107–111 Smith, K. E., Boules, M., Williams, K., Fauq, A. H., and Richelson, E. (2011) The role of NTS2 in the development of tolerance to NT69L in mouse models for hypothermia and thermal analgesia. Behav. Brain Res. 224, 344 –349 MacLellan, C. L., Davies, L. M., Fingas, M. S., and Colbourne, F. (2006) The influence of hypothermia on outcome after intracerebral hemorrhage in rats. Stroke 37, 1266 –1270 Schubert, G. A., Poli, S., Mendelowitsch, A., Schilling, L., and Thome, C. (2008) Hypothermia reduces early hypoperfusion and metabolic alterations during the acute phase of massive subarachnoid hemorrhage: a laser-Doppler-flowmetry and microdialysis study in rats. J. Neurotrauma 25, 539 –548 Torok, E., Klopotowski, M., Trabold, R., Thal, S. C., Plesnila, N., and Scholler, K. (2009) Mild hypothermia (33 C) reduces intracranial hypertension and improves functional outcome after subarachnoid hemorrhage in rats. Neurosurgery 65, 352– 359; discussion 359 Sun, J., LI, J., Deveau, T., Kern, Q., Dix, T. Yu, S.P., Wei, L. (2011) Protective effect of chemical hypothermia in hemoorrhage stroke. Society for Neuroscience Annual Meeting, Session 787.10, poster DD19 (abstr.) Ohta, H., Terao, Y., Shintani, Y., and Kiyota, Y. (2007) Therapeutic time window of post-ischemic mild hypothermia and the gene expression associated with the neuroprotection in rat focal cerebral ischemia. Neurosci. Res. 57, 424 –433 Feigin, V., Anderson, N., Gunn, A., Rodgers, A., and Anderson, C. (2003) The emerging role of therapeutic hypothermia in acute stroke. Lancet Neurol. 2, 529 Feigin, V. L., Anderson, C. S., Rodgers, A., Anderson, N. E., and Gunn, A. J. (2002) The emerging role of induced hypothermia in the management of acute stroke. J. Clin. Neurosci. 9, 502–507 Den Hertog, H. M., van der Worp, H. B., Tseng, M. C., and Dippel, D. W. (2009) Cooling therapy for acute stroke. Cochrane Database Syst. Rev. 21, CD001247 Tang, X. N., and Yenari, M. A. (2010) Hypothermia as a cytoprotective strategy in ischemic tissue injury. Ageing Res. Rev. 9, 61–68


Received for publication January 5, 2012. Accepted for publication March 12, 2012.

CHOI ET AL.