A novel stroke therapy of pharmacologically induced hypothermia after focal cerebral ischemia in mice

Abstract

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.

Figure 1.

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

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. ABS-201 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 ≥6 h. *P < 0.05 vs. saline control; n ≥ 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.

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 μl, 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.

Figure 4.

ABS-201 reduced ischemia-induced 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 μm (A, B); 20 μm (C, D).

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).

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 μl, i.c.v. injection in each animal)-treated groups. Scale bars = 20 μm (A, D); 10 μm (B, C). *P < 0.05, **P < 0.01; n = 4/group.

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.

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.