Dexmedetomidine post-treatment induces neuroprotection via activation of extracellular signal-regulated kinase in rats with subarachnoid haemorrhage

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1 ritish Journal of naesthesia, (): 9 () doi:.9/bja/aev59 Neurosciences and Neuroanaesthesia NEUROSIENES N NEURONESTHESI exmedetomidine post-treatment induces neuroprotection via activation of extracellular signal-regulated kinase in rats with subarachnoid haemorrhage Y. Wang,, R. Han, and Z. Zuo, epartment of nesthesiology, University of Virginia, harlottesville, V 9, US, and epartment of naesthesiology, eijing Tian Tan Hospital, apital Medical University, eijing 5, hina orresponding authors. zzc@virginia.edu or ruquan.han@yahoo.com bstract ackground: exmedetomidine, a sedative agent, provides neuroprotection when administered during or before brain ischaemia. This study was designed to determine whether dexmedetomidine post-treatment induces neuroprotection against subarachnoid haemorrhage (SH) and the mechanisms for this effect. Methods: Subarachnoid haemorrhage was induced by endovascular perforation to the junction of the right middle and anterior cerebral arteries in adult rats. exmedetomidine was applied immediately or h after onset of SH. Neurological outcome was evaluated days after SH. Right frontal cortex area was harvested h after SH for western blotting. Results: Subarachnoid haemorrhage reduced neurological scores and increased brain oedema and blood brain barrier permeability. These effects were attenuated by dexmedetomidine post-treatment. Neuroprotection by dexmedetomidine was abolished by P995, an inhibitor of extracellular signal-regulated kinase (ERK) activation. Phospho-ERK, the activated form of ERK, was increased by dexmedetomidine; this activation was inhibited by P995. onclusions: exmedetomidine post-treatment provides neuroprotection against SH. This effect appears to be mediated by ERK. Key words: dexmedetomidine; extracellular signal-regulated kinase; post-treatment; subarachnoid haemorrhage Subarachnoid haemorrhage (SH) is a devastating disease that accounts for 5% of all strokes. bout 5% of SHs are caused by intracranial aneurysm rupture that often occurs in - to - yr-old patients, a very productive period of human life. lthough the incidence of aneurismal SH is low ( incident per persons yr ), >5% of patients will die with aneurysm rupture. Most measures for treating SH are supportive. Interventions are urgently needed to improve neurological outcome after SH. exmedetomidine is a highly selective agonist for α -adrenergic receptors (α Rs) and is used as a sedative agent with analgesic properties. 5 exmedetomidine applied before or during brain ischaemia can provide neuroprotection against ischaemic braininjuryinadultanimals. However, this approach is limited clinically because most brain ischaemic events are not predicted. neuroprotective strategy might be applicable if treatment after brain ischaemia can still provide neuroprotection. ccepted: November, 5 The uthor. Published by Oxford University Press on behalf of the ritish Journal of naesthesia. ll rights reserved. For Permissions, please journals.permissions@oup.com

2 exmedetomidine post-treatment induces neuroprotection 5 Editor s key points Subarachnoid haemorrhage (SH) causes profound neurological deficits and has few effective pharmacological treatments. In a rat model, dexmedetomidine given h after onset of SH attenuated brain oedema, blood brain barrier permeability, and neurological deficits. Inhibition of extracellular signal-regulated kinase prevented neuroprotection by dexmedetomidine, consistent with a role of ERK in its effect. pplication of dexmedetomidine after a traumatic insult provided protection to hippocampal slices in vitro. However, dexmedetomidine post-treatment-induced neuroprotection in vivo has not been shown. lso, it is not known whether dexmedetomidine is neuroprotective against SH. We hypothesized that dexmedetomidine post-treatment provides neuroprotection against SH. Given that extracellular signal-regulated kinase (ERK) is involved in neuroprotection against ischaemic brain injury, we further hypothesized that dexmedetomidine post-treatment-induced neuroprotection in SH is mediated by activation of ERK. These hypotheses were tested in an endovascular perforation model of SH in rats. Methods The animal protocol was approved by the Institutional nimal are and Use ommittee of the University of Virginia (harlottesville, V, US). ll animal experiments were carried out in accordance with the National Institutes of Health Guide for the are and Use of Laboratory nimals (National Institutes of Health publication no. -, revised in ). Efforts were made to minimize the number of animals used and suffering of animals. This study is reported in accordance with nimal Research: Reporting In Vivo Experiments. Experimental protocols In Experiment, -month-old male Sprague awley rats weighing 7 g (harles River Laboratories Inc., Wilmington, M, US) were randomly assigned to four groups, as follows: sham +saline, sham+dexmedetomidine, SH+saline, and SH+dexmedetomidine. Rats received dexmedetomidine or saline immediately and h after the onset of SH. were evaluated at and h after SH (n=). Subarachnoid haemorrhage grades, brain water content (n=), and Evans blue content in brain tissue (n=) were evaluated at h after SH. Evans blue extravasation was performed to reflect blood brain barrier () permeability. To determine whether delayed application of dexmedetomidine would provide neuroprotection, two groups of rats were studied, as follows: SH+saline and SH+dexmedetomidine with application of dexmedetomidine at and h after onset of SH. We delayed the first dose of dexmedetomidine for h to increase the translational potential of our findings because the majority of patients with SH experience a delay in treatment. exmedetomidine ( µg ml ; catalogue no. 9- -; Hospira, Lake Forest, IL, US) was prepared freshly each day in normal saline. exmedetomidine 5 µg kg or the same volume of saline was injected i.p. This dose is larger than that used in humans (usually µg kg i.v. as the loading dose for sedation) but is commonly used in rats. 5 This dose 5 often induces light sedation in rats. In Experiment, the groups were the same as for Experiment. exmedetomidine or saline was injected immediately after SH was established. Right frontal cortex area (Fr) was harvested h after SH to measure the content of phospho-erk by western blotting (n=). In Experiment, rats were randomly assigned to five groups, as follows: sham, SH, SH+P959, SH+dexmedetomidine, and SH+P959+dexmedetomidine. exmedetomidine was given immediately and h after the onset of SH. The ERK inhibitor P959 (P) at mg kg was injected min before dexmedetomidine application. 7 P959 solution (.5 mg ml ) was prepared (Sigma-ldrich o., Louis, MO, US) in normal saline containing % dimethyl sulfoxide (MSO; Fisher Scientific, Fair Lawn, NJ, US). Rats that did not require P959 injection received injection of the vehicle for P959. were evaluated at and h after SH (n=). The SH grades, brain water content (n=), and permeability (n=) were evaluated at h after SH. Right Fr was harvested at h after SH to quantify phospho-erk content by western blotting (n=). Subarachnoid haemorrhage model Subarachnoid haemorrhage was induced by endovascular perforation as previously described by Garcia and colleagues. riefly, rats were anaesthetized with % isoflurane, tracheally intubated with a -gauge catheter and mechanically ventilated. The animal was placed on a heat pad (Physitemp Instruments Inc., lifton, NJ, US) to maintain body temperature at 7. Subarachnoid haemorrhage was achieved by advancing a sharp monofilament nylon suture (eijing inontech o. Ltd, eijing, hina) into the right internal carotid artery via the external carotid artery. The suture was pushed 5 mm further after resistance was felt in order to perforate the bifurcation of the anterior and middle cerebral arteries. The suture was then withdrawn and the blood flow in the internal carotid artery returned to produce SH. Sham-operated rats underwent the same procedure except that the suture was withdrawn after feeling resistance. Heart rate and pulse oximeter oxygen saturation were monitored continuously and non-invasively using a MouseOX Murine Plus Oximeter System (Starr Life Sciences orporation, Oakmont, P, US). nimals received infiltration to the surgical wound with.5% bupivacaine. Neurological deficits were evaluated and h after SH using an -point scoring system as described by Garcia and colleagues. 9 The modified assessment consisted of six tests, including spontaneous activity, spontaneous movement of four limbs, forepaw outstretching, climbing, body proprioception, and response to whisker stimulation ( points). Subarachnoid haemorrhage grade n -point SH severity grading system described previously was used. The basal cistern was divided into six segments that were scored from to according to the amount of subarachnoid blood clot. total score was calculated by adding the scores from six segments ( points). nimals receiving a score were excluded from the study. ssessment of brain water content rain water content was examined to assess the degree of brain oedema. t h after SH, brains were quickly separated into left and right cerebral hemispheres, cerebellum, and brainstem

3 Wang et al. and weighed (wet weight). rain tissue blocks were dried in an oven at for 7 h and weighed again (dry weight). The percentage of water content was calculated as follows: [(wet weight dry weight)/wet weight] %. Measurement of blood brain barrier permeability lood brain barrier integrity was evaluated by Evans blue extravasation. t h after SH, Evans blue dye (% in saline, 5 ml kg ; Sigma ldrich) was injected via the right femoral vein and allowed to circulate for min. Under anaesthesia, rats were transcardially perfused with saline to remove the intravascular dye. rains were then removed and divided into left and right cerebral hemispheres, cerebellum, and brainstem. The brain samples were weighed and homogenized in ml of 5% trichloroacetic acid (Sigma-ldrich), incubated overnight at, and centrifuged at g for min. The amount of Evans blue in the supernatant was quantified by spectrophotometry at nm. The results were expressed as micrograms per gram of brain tissue wet weight. Immunoblot analysis Rats were killed by deep isoflurane anaesthesia and transcardially perfused with normal saline at h after SH. The right Fr (perforation side) between bregma + and mm was harvested for western immunoblot analysis. Tissues were homogenized in RIP buffer (5 mm Tris Hl, ph 7., 5 mm Nal, % NP-, % sodium deoxycholate, and.% SS; Thermo Scientific, Rockford, IL, US) with protease inhibitors ( mg ml aprotinin, 5 mg ml peptastin, 5 mg ml leupeptin, and mm phenylmethanesulfonylfluoride; catalogue no. M5V; Sigma-ldrich) and phosphatase inhibitors (PhosSTOP; Roche iagnostics GmbH, Mannheim, Germany) on ice as described. Homogenates were centrifuged at at g for 5 min, and the supernatant was used for immunoblotting. Equivalent total protein amounts ( µg) were loaded into each lane of preformed SS-PGE gels (%; io-rad Laboratories Inc., Hercules,, US). fter gel electrophoresis, proteins were transferred onto a nitrocellulose membrane (io-rad Laboratories Inc.) and shaken with blocking buffer (Thermo Scientific) for h at room temperature. The following primary antibodies were incubated with the membrane under gentle agitation at overnight: mouse anti-phospho-erk antibody (:, catalogue no. 7; Santa ruz iotechnology, Santa ruz,, US), rabbit anti-erk antibody (:, catalogue no. F; Santa ruz iotechnology) and rabbit anti-β-actin antibody (:, catalogue nuo. 97S; ell Signaling Technology, anvers, M, US). Secondary horseradish peroxidase-conjugated goat anti-mouse antibody (:5, catalogue no. E; Santa ruz iotechnology) or goat anti-rabbit antibody (:5, catalogue no. H; Santa ruz iotechnology) was used. fter visualization with enhanced chemiluminescence, quantitative analysis of the protein bands was performed using an Image-Quant 5. GE Healthcare ensitometer (GE Healthcare, Sunnyvale,, US). ensities of phospho-erk protein bands were normalized to those of ERK in the same sample to control for errors in protein sample loading and transferring. Statistical analysis Parametrical data are presented as the mean (S). Results of brain water content, Evans blue extravasation and immunoblotting were analysed by one-way analysis of variance, followed by Tukey s test. and SH grade were analysed by one-way analysis of variance on ranks, followed by Tukey s test. value of P.5 was accepted as significant. ll statistical analyses were performed using SigmaStat (SYSTT Software Inc., Point Richmond,, US). Results exmedetomidine neuroprotection Three rats had SH grades in SH and SH+dexmedetomidine post-treatment groups in Experiment, and these animals were excluded in the final data analysis. Five and four rats in the SH and SH+dexmedetomidine post-treatment groups, respectively, died within h after onset of SH, corresponding to and % for these groups. No rats in the sham-operated groups died within the h observation time. Similar mortality rates were observed and numbers of rats excluded from the final analysis owing to low SH grades occurring in the SH and sham-operated groups in the other sets of experiments. The body weights of rats in the sham-operated, SH, and SH +dexmedetomidine post-treatment groups were (7), (), and (9) g (P>.5), respectively, before surgery and (), (), and (5) g, respectively, for sham-operated, SH, and SH+dexmedetomidine post-treatment groups at h after SH surgery. The body weights of rats in the SH group were significantly decreased after SH compared with their weights before SH (P=.). Subarachnoid haemorrhage grades were similar among the SH groups with or without dexmedetomidine post-treatment. were significantly reduced by SH [median (5th 75th percentile): (, 5) compared with (, ) of sham-operated animals at h after onset of SH, P.]. This reduction was reversed by dexmedetomidine, with the first dose applied immediately after onset of SH [ (5, 7), P=. compared with SH only]. This pattern existed when assessment of neurological scores was performed or h after the onset of SH (Fig. ). Likewise, application of dexmedetomidine at h after onset of SH (post-treatment) improved neurological outcome after SH (Fig. ). onsistent with these results, the right cerebral hemisphere in the SH group contained more water than that in the sham-operated group (water content:.7 (.) and 7.5 (.)% in the SH group and sham-operated group, respectively, P.). This increase was attenuated by dexmedetomidine post-treatment [79.5 (.)%, P=.7 compared with the SH group; Fig. ). Likewise, Evans blue content in brain tissue, which reflects the permeability of the, was increased in the right cerebral hemisphere. This increase was also attenuated by dexmedetomidine post-treatment (Fig. ). These results suggest that dexmedetomidine post-treatment provides neuroprotection after SH. Interestingly, SH from a right cerebral vessel also increased Evans blue content in the left cerebral hemisphere, cerebellum, and brainstem. This increase in the left cerebral hemisphere was significantly attenuated by dexmedetomidine applied immediately after SH (Fig. ). These results suggest that the effects of SH on the brain can be diffuse. Role of extracellular signal-regulated kinase in dexmedetomidine neuroprotection Phospho-ERK immunoreactivity in the right Fr was reduced by SH, but this was significantly increased by dexmedetomidine post-treatment. This increase was abolished by P995, an ERK inhibitor (Fig. and ). These results suggest that dexmedetomidine post-treatment can activate ERK in the brain after SH.

4 exmedetomidine post-treatment induces neuroprotection 7 SH grades h after SH h after SH ( h delay) Fig exmedetomidine post-treatment-induced neuroprotection. dult male rats had SH induced by endovascular perforation of the junction of right middle and anterior cerebral arteries. They were treated with dexmedetomidine, with the first dose given immediately or h after onset of SH. () representative brain images. () Subarachnoid haemorrhage grades. () at h after onset of SH. () at h after onset of SH. Inside boxes: 5 75% interval, including the median; n= for the first four groups and n=forthelasttwogroups.p.5 compared with sham-operated animals. ^P.5 compared with SH-only group. ex, dexmedetomidine; SH, subarachnoid haemorrhage. There was no difference in the SH grades between SH groups treated with or without dexmedetomidine and P995. onsistent with these results, neurological scores were decreased by SH when assessment was performed or h after SH, which was attenuated by dexmedetomidine. P995 blocked the effect of dexmedetomidine (Fig. and ). Water content in the right cerebral hemisphere was increased by SH, and this was attenuated by dexmedetomidine. P995 abolished the effect of dexmedetomidine (Fig. 5). Similar effects occurred in Evans blue content in the right cerebral hemisphere. Evans blue content in the left cerebral hemisphere, cerebellum, and brainstem was increased. This increase was attenuated by dexmedetomidine in the left cerebral hemisphere, which was prevented by P995 (Fig. ). P995 did not affect the neurological scores, water, and Evans blue content in the brain of animals with SH only (Figs ). These results suggest that the effects of dexmedetomidine are mediated by ERK. iscussion Our results show that SH impairs neurological function and causes weight loss. exmedetomidine post-treatment improved the neurological outcome after SH, and also reduced water content and permeability. These effects were evident even when the first dose of dexmedetomidine was given h after the onset of SH. These results provide initial in vivo evidence for dexmedetomidine post-treatment-induced neuroprotection against SH. There are three studies that have investigated the neuroprotective effects of dexmedetomidine when administered after injury. exmedetomidine applied h after transient global cerebral ischaemia did not provide neuroprotection in adult gerbils. 9 In contrast, application of dexmedetomidine h, but not h, after a traumatic insult provided neuroprotection in mouse brain slices. In addition, incubation of mouse hippocampal slices with dexmedetomidine for h at h after oxygen and glucose deprivation reduced cell death. 5 Our results show that dexmedetomidine applied h after SH was neuroprotective. lthough the reason for the failure to show neuroprotection in the gerbil study is not known, the delay in application of dexmedetomidine or differences between SH and the ischaemia model used could have contributed to it. exmedetomidine post-treatment-induced neuroprotection against SH appears to be mediated by ERK. exmedetomidine post-treatment increased the activated ERK. P995, an ERK inhibitor, inhibited this increase and also blocked the effects of dexmedetomidine on neurological scores, water content, and Evans blue content in brain. These results suggest a critical role of ERK in the dexmedetomidine post-treatment effects. Extracellular signal-regulated kinase has been suggested to be a protein kinase essential for cell survival. exmedetomidine might activate ERK to mediate its neuroprotection against ischaemic brain injury when it is applied during brain ischaemia in rats or after a traumatic injury in mouse hippocampal slices. lso, dexmedetomidine can activate ERK. 7 Thus, ERK appears to be an important intracellular signaling molecule for dexmedetomidineinduced neuroprotection. Two pathways have been described for dexmedetomidine to activate ERK. The first is via activation of α Rs, which induces release of epidermal growth factor or epidermal growth factorlike growth factor, and then activates/phosphorylates ERK. 9 The second pathway is α R independent. exmedetomidine could activate imidazoline I receptors that then increase protein

5 Wang et al. 7 Left cerebral hemisphere 7 Right cerebral hemisphere erebellum rainstem 7 7 ( h delay) Fig exmedetomidine post-treatment-induced reduction of brain oedema after SH. dult male rats had SH induced by endovascular perforation of the junction of right middle and anterior cerebral arteries. They were treated with dexmedetomidine, with the first dose given immediately or h after onset of SH. () Water content in left cerebral hemisphere. () Water content in right cerebral hemisphere. () Water content in cerebellum. () Water content in brainstem. Results are mean (S); n=. P.5 compared with sham-operated animals. ^P.5 compared with SH-only group. ex, dexmedetomidine; SH, subarachnoid haemorrhage. Left cerebral hemisphere Right cerebral hemisphere erebellum rainstem ( h delay) Fig exmedetomidine post-treatment-induced reduction of blood brain barrier permeability after SH. dult male rats had SH induced by endovascular perforation of the junction of right middle and anterior cerebral arteries. They were treated with dexmedetomidine, with the first dose given immediately or h after the onset of SH. () Evans blue content in left cerebral hemisphere. () Evans blue content in right cerebral hemisphere. () Evans blue content in cerebellum. () Evans blue content in brainstem. Results are mean (S); n= for the first four groups and n= for the last two groups. P.5 compared with sham-operated animals. ^P.5 compared with SH-only group. ex, dexmedetomidine; SH, subarachnoid haemorrhage.

6 exmedetomidine post-treatment induces neuroprotection 9 Sham-sal P-ERK Phospho-ERK abundance Phospho-ERK abundance ERK b-actin 5 Sham-sal P-ERK ERK b-actin 5 dex # SH sal SH dex P-dex sal P SH-P-dex SH-P SH grades SH-P SH-P-dex h after SH h after SH # # Fig exmedetomidine post-treatment-induced activation of ERK and neuroprotection after SH. dult male rats had SH induced by endovascular perforation of the junction of right middle and anterior cerebral arteries. They were treated with dexmedetomidine, with the first dose given immediately after the onset of SH. () Phospho-ERK immunoreactivity in the frontal cortex area. () Phospho-ERK immunoreactivity in the frontal cortex area for animals treated with P995 at min before dexmedetomidine application. () Subarachnoid haemorrhage grades. (). Results are mean (S) [n= for panels () and ()] or in box plot [n= for panels () and()]. P.5 compared with sham-operated animals. ^P.5 compared with SH-only group. #P.5 compared with SH +dexmedetomidine group. ex, dexmedetomidine; ERK, extracellular signal-regulated kinase; P, P995; SH, subarachnoid haemorrhage; Sal, saline. kinase activity to activate ERK. 7 These two pathways might work together to enhance activation of ERK by dexmedetomidine. Subarachnoid haemorrhage induces cerebral oedema by various mechanisms, such as autoregulatory breakdown in brain blood vessels because of hypertension, neuroinflammation, focal brain ischaemia, and increased intracellular water attributable to hyponatraemia. Many of these mechanisms result in increased permeability as demonstrated in our study. exmedetomidine might inhibit these mechanisms via its neuroprotective effects, and might also work through α R to control the permeability directly. These effects should contribute to dexmedetomidine-induced reduction of brain oedema after SH as observed in the present study. Our study could have significant translational implications. Subarachnoid haemorrhage is not uncommon and has a high mortality. If our findings in rats are confirmed in humans, translation of our finding to humans could easily be achieved. In addition, dexmedetomidine can be used to sedate patients with SH in the intensive care unit. Thus, dexmedetomidine can be used for two purposes in this situation. Our study has limitations. We did not perform a dose response study and selected a dose that was protective against ischaemic stroke. Our SH model simulates the clinical situation of intracranial aneurysm rupture. However, it is a difficult model and requires a large number of animals per group. ose response studies should be performed in the future to provide better characterization of the effects of dexmedetomidine on SH. lso, mechanisms other than ERK activation could play a role in dexmedetomidine post-treatment against SH. α -drenergic receptors can regulate neurotransmitter release, but dexmedetomidine does not affect brain ischaemia reperfusioninduced glutamate release. 5 In addition, our study showed that P995 abolished dexmedetomidine post-treatment-induced neuroprotection. Thus, the effects of dexmedetomidine

7 9 Wang et al. 7 7 Left cerebral hemisphere 7 7 Right cerebral hemisphere # erebellum rainstem SH-P SH-P-dex Fig 5 ttenuation of dexmedetomidine post-treatment effects on brain oedema after SH by extracellular signal-regulated kinase inhibition. dult male rats had SH induced by endovascular perforation of the junction of right middle and anterior cerebral arteries. They were treated with dexmedetomidine, with the first dose given immediatelyafteronset of SH. P995 was given min before dexmedetomidine application. () Water content in left cerebralhemisphere. () Watercontent in right cerebral hemisphere. () Water content in cerebellum. () Water content in brainstem. Results are mean (S); n=. P.5 compared with sham-operated animals. ^P.5 comparedwith SH-onlygroup. #P.5 comparedwith SH+dexmedetomidine group. ex, dexmedetomidine; P, P995; SH, subarachnoid haemorrhage. 5 Left cerebral hemisphere 5 Right hemisphere # erebellum rainstem SH-P SH-P-dex Fig ttenuation of dexmedetomidine post-treatment effects on blood brain barrier permeability after SH by extracellular signal-regulated kinase inhibition. dult male rats had SH induced by endovascular perforation of the junction of right middle and anterior cerebral arteries. They were treated with dexmedetomidine, with the first dose given immediately after the onset of SH. P995 was given min before dexmedetomidine application. () Evans blue content in left cerebral hemisphere. () Evans blue content in right cerebral hemisphere. () Evans blue content in cerebellum. () Evans blue content in brainstem. Results are mean (S); n=. P.5 compared with sham-operated animals. ^P.5 compared with SH-only group. ex, dexmedetomidine; SH, subarachnoid haemorrhage.

8 exmedetomidine post-treatment induces neuroprotection 9 post-treatment on glutamate release might not be important for the effects observed in our study and have not been determined. Finally, we studied only male rats; therefore, potential sex differences were not determined. In summary, our results suggest that dexmedetomidine posttreatment induces neuroprotection against SH in rats, mediated by ERK activation. uthors contributions onception of the project: Z.Z. esign of the study: Y.W., R.H., Z.Z. Performance of the experiments: Y.W. Initial data analysis and drafting of the Methods section: Y.W. Final data analysis and writing of the manuscript: Z.Z. eclaration of interest None declared. Funding National Institutes of Health, ethesda, M, US (R GM9 to Z.Z.); International naesthesia Research Society, leveland, OH, US (7 Frontiers in naesthesia Research ward to Z. 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