Baclofen mediates neuroprotection on hippocampal CA1 pyramidal cells through the regulation of autophagy under chronic cerebral hypoperfusion

GABA receptors play an important role in ischemic brain injury. Studies have indicated that autophagy is closely related to neurodegenerative diseases. However, during chronic cerebral hypoperfusion, the changes of autophagy in the hippocampal CA1 area, the correlation between GABA receptors and autophagy, and their influences on hippocampal neuronal apoptosis have not been well established. Here, we found that chronic cerebral hypoperfusion resulted in rat hippocampal atrophy, neuronal apoptosis, enhancement and redistribution of autophagy, down-regulation of Bcl-2/Bax ratio, elevation of cleaved caspase-3 levels, reduction of surface expression of GABAA receptor α1 subunit and an increase in surface and mitochondrial expression of connexin 43 (CX43) and CX36. Chronic administration of GABAB receptors agonist baclofen significantly alleviated neuronal damage. Meanwhile, baclofen could up-regulate the ratio of Bcl-2/Bax and increase the activation of Akt, GSK-3β and ERK which suppressed cytodestructive autophagy. The study also provided evidence that baclofen could attenuate the decrease in surface expression of GABAA receptor α1 subunit, and down-regulate surface and mitochondrial expression of CX43 and CX36, which might enhance protective autophagy. The current findings suggested that, under chronic cerebral hypoperfusion, the effects of GABAB receptors activation on autophagy regulation could reverse neuronal damage.


Baclofen inhibited autophagy of hippocampal CA1 area under chronic cerebral hypoperfusion.
To investigate whether autophagy is involved in the neuroprotection of GABA B receptors activation under chronic cerebral hypoperfusion, we firstly examined the activation of autophagy in cortex and hippocampal CA1 area. As shown in Fig. 2, five weeks after induction of hypoperfusion, the LC3 immunoreactivity was slightly but significantly increased in cortex. However, in hippocampal CA1 area, we observed a robust increase in the LC3 immunoreactivity, which was consistent with the protein expression of LC3-II. Hippocampus is the area that displays the most characteristic neuropathological damage in neurodegenerative disorders and hippocampal CA1 area is one of the brain regions most sensitive to ischemia. Thus, in the follow-up experiments, we further investigated the role of baclofen in autophagy of hippocampal CA1 area under chronic cerebral hypoperfusion. Our results revealed that LC3 immunoreactivity was low in the sham-operated group, and uniformly distributed along CA1 pyramidal cell axons. Chronic cerebral hypoperfusion led to a redistribution of LC3 immunoreactivity from CA1 pyramidal cell axons to abundant punctate structures in the cell body. Chronic treatment with baclofen significantly decreased the LC3 immunoreactivity and prevented LC3 redistribution (Fig. 3a,b). To further confirm that baclofen could suppress chronic cerebral hypoperfusion-induced autophagy, we identified the expression of protein markers characteristic for autophagy, such as p-mTOR, Beclin 1, atg5, atg7 and LC3-II in the hippocampal CA1 area with Western blot analyses. Our results showed that, five weeks after induction of hypoperfusion, p-mTOR was significantly decreased, and LC3-II, Beclin 1, atg5 and atg7 were significantly increased. Baclofen could reverse the changes of these proteins expression. Treatment with baclofen at 12.5 mg/kg and 25 mg/kg in sham-operated rats did not change the expression of p-mTOR, LC3-II, Beclin 1, atg5 and atg7 compared with sham-operated rats (Fig. 4a,b).

Baclofen diminished chronic hypoperfusion-induced neuronal apoptosis.
In the present study, H&E and TUNEL staining was used to examine the influence of baclofen on degenerative changes of hippocampal CA1 area. Five weeks after induction of hypoperfusion, hippocampal atrophy and significant neuronal loss in hippocampal CA1 area were detected. Chronic treatment with baclofen markedly diminished hippocampal atrophy and neuronal loss in hippocampal CA1 area (Fig. 5). Besides, TUNEL-stained positive cells were significantly increased in hippocampal CA1 area in 2VO rats. Treatment with baclofen at 12.5 mg/kg and 25 mg/kg in 2VO rats significantly reduced the number of TUNEL-stained positive cells (Fig. 6a,b). Furthermore, we found that, five weeks after induction of hypoperfusion, the expression of Bax was not significantly changed compared with sham-operated rats. Treatment with baclofen at 25 mg/kg in 2VO rats significantly enhanced the expression of Bax. Treatment with baclofen at 12.5 mg/kg and 25 mg/kg in sham-operated rats did not change the expression of Bax compared with sham-operated rats. Besides, five weeks after induction of hypoperfusion, the expression of Bcl-2 was significantly decreased, treatment with baclofen recovered Bcl-2 expression. Treatment with baclofen at 12.5 mg mg/kg and 25 mg/kg in sham-operated rats significantly increased the expression of Bcl-2 compared with sham-operated rats. Our results revealed that chronic cerebral hypoperfusion significantly decreased the ratio of Bcl-2/Bax in the hippocampal CA1 region, and baclofen could up-regulate Bcl-2/Bax ratio (Fig. 7a,b). We also found that, five weeks after induction of hypoperfusion, the expression of pro-caspase-3 was significantly decreased compared with sham-operated rats, and treatment with baclofen recovered pro-caspase-3 expression (Fig. 7c). We further evaluated the levels of cleaved caspase-3 (an activated form of caspase-3) in each group. Our results showed that hypoperfusion resulted in a significant increase in cleaved caspase-3 levels of hippocampal CA1 cells, chronic treatment with baclofen significantly reduced cleaved caspase-3 levels (Fig. 7d). In sham group, the LC3 immunoreactivity was very low. Treatment with baclofen in sham group had no significant change in the LC3 immunoreactivity. In the brain slice model of OGD-Rep injury, the LC3 immunoreactivity was robustly elevated compared with sham group, whereas in the OGD-Rep+ baclofen (100 μ M) group, the LC3 immunoreactivity was declined towards basal levels (n = 4 in each group). (b) Quantitative analysis of the LC3 immunoreactivity. (c,d) The protein levels of LC3 II were significantly increased in OGD-Rep group, but baclofen markedly alleviated excessive autophagy. OGD-Rep injury given rise to a significant increase in cleaved caspase-3 levels, baclofen significantly decreased cleaved caspase-3 levels (n = 4 in each group). * P < 0.05 and ** P < 0.01 vs sham group; # P < 0.05 and ## P < 0.01 vs OGD-Rep group.
Scientific RepoRts | 5:14474 | DOi: 10.1038/srep14474 Baclofen could enhance the phosphorylation of protein kinase B (Akt) (Ser473), glycogen synthase kinase 3β (GSK-3β) (Ser-9) and extracellular regulated protein kinases 1/2 (ERK1/2). In order to further explore the possible mechanisms of association between GABA B receptors and autophagy in chronic cerebral hypoperfusion, we detected the phosphorylation as well as total level of Akt, GSK-3β , and ERK1/2 in the hippocampal CA1 area with Western blot analyses. Our results showed that p-Akt and p-GSK-3β were not significantly changed in the hippocampal CA1 region of 2VO rats. However, hypoperfusion caused a slight but significant increase in p-ERK1/2. Chronic treatment with baclofen significantly enhanced the phosphorylation of Akt, GSK-3β and ERK1/2. There were no significant changes in the expression of total Akt, GSK-3β , and ERK1/2 in each group (Fig. 8a-c).
Baclofen could attenuate 2VO-induced reduction of GABA A receptor α1 subunit surface expression. In this study, we identified the surface expression of GABA A receptors in the hippocampal CA1 area with Western blot analyses. No contamination with cytosolic protein was observed as GAPDH was not seen by Western blot in these samples (data not shown). Our results showed that, five weeks after induction of hypoperfusion, the surface expression of GABA A receptor α 1 subunit was significantly decreased, and intracellular expression of GABA A receptor α 1 subunit was significantly increased. Baclofen could attenuate 2VO-induced reduction of GABA A receptor α 1 subunit surface expression (Fig. 9a). Besides, treatment with baclofen (25 mg/kg) in sham-operated rats accelerated the decrease in the population of surface GABA A receptor α 1 subunit, and the increase in the population of intracellular GABA A receptor α 1 subunit (Fig. 9a). Five weeks after induction of hypoperfusion, the LC3 immunoreactivity was slightly but significantly increased in cortex, however, a robust increase in the LC3 immunoreactivity was observed in hippocampal CA1 area (n = 4 in each group). (b) Quantitative analysis of the LC3 immunoreactivity. (c) The protein expression of LC3 II in cortex and hippocampal CA1 area (n = 4 in each group). Blots shown have been cropped to fit space requirements and run under the same experimental conditions. *P < 0.05 vs sham-operated rats (cortex), ## P < 0.01 vs sham-operated rats (hippocampal CA1 area). Baclofen could down-regulate the surface and mitochondrial expression of CX43 and CX36 under chronic cerebral hypoperfusion. Our results showed that, five weeks after induction of hypoperfusion, the surface and mitochondrial expression of CX43 and CX36 were significantly increased. Baclofen could reduce the surface and mitochondrial expression of CX43 and CX36 in 2VO rats; treatment with baclofen in sham-operated rats did not significantly change CX43 and CX36 surface and In the sham-operated group, LC3 immunoreactivity was low and uniformly distributed along CA1 pyramidal cell axons, chronic cerebral hypoperfusion led to a distribution of LC3 immunoreactivity from CA1 pyramidal cell axons to abundant punctate structures in the cell body. Chronic treatment with baclofen significantly decreased the LC3 immunoreactivity and prevented LC3 redistribution. Treatment with baclofen at 12.5 mg/kg and 25 mg/kg in sham-operated rats had no significant effect on the LC3 immunoreactivity. (b) Quantitative analysis of the LC3 immunoreactivity (n = 4 in each group). ** P < 0.01 vs sham-operated rats; ## P < 0.01 vs 2VO rats.

Discussion
In the present study, we demonstrated for the first time that chronic treatment with baclofen markedly diminished hippocampal atrophy and neuronal apoptosis in hippocampal CA1 area via the regulation of autophagy in chronic cerebral hypoperfusion in rats.
Autophagy in the CNS is a double-edged sword. Proper course of autophagy in the CNS determines the maintenance of cellular homeostasis, providing cytoprotection against stress-induced apoptosis. However, extensive autophagy destroys large proportions of the cytosol and organelles that, beyond a certain threshold, would cause irreversible cellular atrophy and trigger either apoptosis or necrotic cell (a-c) Five weeks after induction of hypoperfusion, p-mTOR was significantly decreased, and LC3-II, Beclin 1, atg5 and atg7 were significantly increased, and baclofen could reverse the changes of these proteins expression. Treatment with baclofen at 12.5 mg/kg and 25 mg/kg in sham-operated rats did not change the expression of LC3-II, mTOR, p-mTOR, Beclin 1, atg5 and atg7 compared with sham-operated rats (n = 4 in each group). Blots shown have been cropped to fit space requirements and run under the same experimental conditions. * P < 0.05 and ** P < 0.01 vs sham-operated rats; ## P < 0.01 vs 2VO rats. death 53 . Many studies have reported that uncontrolled excessive induction of autophagy in response to ischemia injury may contribute to "autophagic cell death", which is introduced to describe a form of programmed cell death morphologically distinct from apoptosis and characterized by the presence of intense autophagy 54,55 , and the inhibition of excessive autophagy can attenuate cerebral ischemia-associated neuronal damage [11][12][13][14][15][16] . Consistent with these studies, our present study found that baclofen could attenuate 2VO-induced increase in autophagy in hippocampal CA1 area. There is now mounting evidence that autophagy and apoptosis may share common molecular inducers and regulatory mechanism. It has been shown that Atg5 enhances caspase-dependent death though interacting directly with FADD (Fas-associated via death domain) 53 . Besides, calpain-mediated cleavage of Atg5 promotes cytochrome c release and caspase activation and thus switches autophagy to apoptosis 56 . Furthermore, studies have shown that increased Beclin 1 expression colocalizes with activated caspase-3 after adult focal cerebral ischemia and hypoxia-ischemia 9,57 , and binding of the antiapoptotic protein Bcl-2 to Beclin 1 inhibits autophagy 16,53 . A recent study has reported that Bcl-2 negatively regulates autophagy by inhibiting Bax and Bcl-2 homologous antagonist/killer (Bak) 58 . Our current results revealed that, under chronic cerebral hypoperfusion, baclofen could simultaneously increase the expression of Bcl-2 and Bax (especially for Bcl-2) by promoting the phosphorylation of ERK (as described below) 59,60 , but up-regulate Bcl-2/Bax ratio, which might both inhibit autophagy and down-regulate cleaved caspase-3 53,61,62 . Besides, it has been reported that activation of Akt does not alter the levels of Bax and Bcl-2, but Akt phosphorylation can prevent Bax translocation to mitochondria, which inhibits cytochrome c release 63,64 and may repress autophagy 65 . All of the evidence above suggested that neuroprotection of GABA B receptors activation might be closely related to its role in the regulation of autophagy. It has been reported that neurons can regulate the two opposite downstream effects of autophagy, survival and death, after ischemia 2 . It is very important to investigate the possible mechanism for this.  implicated in autophagy 16,73 . It means that the effects of ERK and Akt activation on the regulation of autophagy may be at least partly involved in ERK/Akt-mediated neuroprotection.
It has been reported that activation of MEK/ERK downstream of AMPK leads to disassembly of mTORC1 and mTORC2, and an increase in Beclin 1 expression 7 . In this report, our results showed that, under chronic cerebral hypoperfusion, treatment with baclofen significantly enhanced the phosphorylation Figure 7. Baclofen attenuated chronic hypoperfusion-induced neuronal apoptosis. (a) Five weeks after induction of hypoperfusion, the expression of Bax was no significant change compared with shamoperated rats. Treatment with baclofen at 25 mg/kg in 2VO rats significantly enhanced the expression of Bax. Treatment with baclofen at 12.5 mg/kg and 25 mg/kg in sham-operated rats did not change the expression of Bax compared with sham-operated rats (n = 4 in each group). (b) Five weeks after induction of hypoperfusion, the expression of Bcl-2 was significantly decreased, treatment with baclofen at 12.5 mg/ kg and 25 mg/kg in 2VO rats significantly increased the expression of Bcl-2. Treatment with baclofen at 12.5 mg/kg and 25 mg/kg in sham-operated rats significantly increased the expression of Bcl-2 compared with sham-operated rats (n = 4 in each group). Our results revealed that chronic cerebral hypoperfusion significantly decreased the ratio of Bcl-2/Bax in the hippocampal CA1 region, and baclofen could upregulate Bcl-2/Bax ratio. (c) Five weeks after induction of hypoperfusion, the expression of pro-caspase-3 was significantly decreased compared with sham-operated rats, and treatment with baclofen recovered procaspase-3 expression (n = 4 in each group). (d) Five weeks after induction of hypoperfusion, the levels of cleaved caspase-3 in hippocampal CA1 cells were significantly increased, chronic treatment with baclofen significantly reduced cleaved caspase-3 levels (n = 4 in each group). Blots shown have been cropped to fit space requirements and run under the same experimental conditions. ** P < 0.01 vs sham-operated rats; # P < 0.05 and ## P < 0.01 vs 2VO rats; • P < 0.05 and •• P < 0.01 vs sham-operated rats.
Scientific RepoRts | 5:14474 | DOi: 10.1038/srep14474 of ERK1/2, but inhibited the expression of Beclin 1. The main reasons for this inconsistent result may be as follows. On the one hand, under chronic cerebral hypoperfusion, baclofen-induced ERK1/2 phosphorylation can increase Beclin 1 expression moderately. On the other hand, baclofen-induced Akt phosphorylation may significantly down-regulate the expression of Beclin 1, since it has been reported that the PI3K/Akt inhibitor LY294002 abrogates the down-regulation effect of melatonin on Beclin-1 expression in rat model of transient focal cerebral ischemia 74 . The combined effect of these was that activation of GABA B receptors reduced Beclin 1 expression and inhibited autophagy under chronic cerebral hypoperfusion. However, treatment with baclofen in sham-operated rats did not significantly change the expression of Beclin 1 compared with sham operated rats. It meant that, under physiological conditions, baclofen-induced ERK1/2 and Akt phosphorylation could keep autophagy in balance. One study has shown that transiently or moderately activated MEK/ERK leads to the inhibition of either mTORC1 or mTORC2 and the moderate increase in Beclin 1 expression, resulting in cytoprotective autophagy 7 . Wang (a-c) p-Akt and p-GSK-3β were not significantly changed in the hippocampal CA1 region of 2VO rats, however, hypoperfusion caused a slight but significant increase in p-ERK1/2. Chronic treatment with baclofen further significantly enhanced the phosphorylation of Akt, GSK-3β and ERK1/2. There was no significant change in the expression of total Akt, GSK-3β , and ERK1/2 (n = 4 in each group). Blots shown have been cropped to fit space requirements and run under the same experimental conditions. * P < 0.05 vs sham-operated rats; # P < 0.05 and ## P < 0.01 vs 2VO rats; • P < 0.05 and •• P < 0.01 vs sham-operated rats.  (a) Five weeks after induction of hypoperfusion, the surface expression of GABA A receptor α 1 subunit was significantly decreased, intracellular expression of GABA A receptor α 1 subunit was significantly increased. Baclofen could attenuate 2VO-induced reduction of GABA A receptor α 1 subunit surface expression; treatment with baclofen (25 mg/kg) in sham-operated rats accelerated the decrease in the surface expression of GABA A receptor α 1 subunit, and the increase in the population of intracellular GABA A receptor α 1 subunit (n = 5 in each group). (b,c) Five weeks after induction of hypoperfusion, the surface and mitochondrial expression of CX43 and CX36 was significantly increased, baclofen could reduce CX43 and CX36 surface and mitochondrial expression in 2VO rats; treatment with baclofen in sham-operated rats did not significantly change CX43 and CX36 surface and mitochondrial expression (n = 4 in each group). Blots shown have been cropped to fit space requirements and run under the same experimental conditions. * P < 0.05 and ** P < 0.01 vs sham-operated rats; # P < 0.05 and ## P < 0.01 vs 2VO rats; • P < 0.05 and •• P < 0.01 vs sham-operated rats.
Many studies have shown that the activated Akt kinase not only suppresses the proapoptotic function of Bax 63,64,76 but also inhibits the activity of hamartin (TSC1) and tuberin (TSC2) protein complex, which reduces the GTPase activity of Ras homolog enriched in brain (Rheb) and leads to activation of mTOR and subsequent inhibition of autophagy 16 . In this report, we found that the activity of Akt was much higher in baclofen-treated rats than that in 2VO and sham-operated rats. We then examined the changes in GSK-3β activity, because Akt was an upstream regulator of GSK-3β phosphorylation which had also been proposed as an intracellular signalling mechanism mediating autophagy. It has been reported that phosphorylation of GSK-3β can be conducive to autophagy repression 52 . Our data revealed that, under chronic cerebral hypoperfusion, baclofen significantly enhanced GSK-3β phosphorylation, which could both increase p-mTOR levels and reduce the expression and function of Beclin 1 77 . A recent study has shown that the inhibition of autophagy via activation of PI3K/Akt pathway has neuroprotective role in transient global ischemia 17 . Together, our observations indicated that baclofen might suppress cytodestructive autophagic activity through Akt-GSK-3β -p-mTOR-Beclin 1 signaling pathway under chronic cerebral hypoperfusion.
In addition, a more recent study has reported that plasma membrane Cx43 and other members of the Cx family contribute to negatively modulate autophagy, which seems independent of their function in intercellular communication and signaling, but requires the physical interaction of autophagy precursors, such as Atg16 52 . Studies have reported that CX43 and CX36 are significantly increased in animal models of unilateral middle cerebral artery occlusion (MCAO), oxygen-glucose deprivation (OGD) 50 , and transient brain ischemia 44,51,78 . Inhibiting the expression and function of CX43 and CX36 may be involved in the neuroprotection 44,50 . Thus, we speculated that, the increased expression of CX43 and CX36 could reduce cytoprotective autophagy and promote neuronal death during ischemia. In the present study, our results showed that, five weeks after induction of hypoperfusion, the surface expression of CX43 and CX36 was significantly increased, and baclofen could reduce CX43 and CX36 surface expression. It meant that, during chronic cerebral hypoperfusion, the up-regulation of CX43 and CX36 surface expression suppressed cytoprotective autophagy, and mediated the spread of pro-death signals that resulted in widespread neuronal demise. Baclofen-induced suppressed the increase in CX43 and CX36 surface expressions, thus helping neurons survive.
Studies have reported that GABA A receptors activation can down-regulate the expression of Cx43 43 and Cx36 44 in the CNS. In the present study, our results revealed that chronic GABA B receptors agonist exposure could attenuate 2VO-induced reduction of the surface expression of GABA A receptors α 1 subunit. However, treatment with baclofen (25 mg/kg) in sham-operated rats accelerated the decrease in the population of surface GABA A receptor α 1 subunit, which was consistent with previous studies that had shown activation of GABA B receptors could reduce GABA A receptors -mediated currents under normal conditions 79,80 . The main reasons why activation of GABA B receptors played different roles in GABA A receptors expression under normal condition and chronic cerebral hypoperfusion may be as follows. A recent study has reported that an increase in the intracellular Ca 2+ concentration can enhance the desensitization of GABA A receptors in the barrel cortex in PRIP-1/2 double-knockout (PRIP-DKO) mice 81 . So we speculated that chronic hypoperfusion-induced increase in the intracellular Ca 2+ concentration could enhance the desensitization of GABA A receptors resulting in the reduction of GABA A receptors surface expression. Previous studies have reported that dose-dependent administration of baclofen depresses Ca 2+ -influx [82][83][84] . Besides, it has been demonstrated that GABA B receptors activation suppresses NMDA receptors -mediated Ca 2+ influx by attenuating the activity of Src in rat four-vessel occlusion (4-VO) ischemic model 31 . Thus, under chronic cerebral hypoperfusion, activation of GABA B receptors might contribute to the resensitization of GABA A receptors and restore GABA A receptors surface expression by depressing Ca 2+ -influx. However, under normal conditions, in order to maintain the balance of excitation and inhibition, the inhibiting effect of GABA B receptors activation on neuronal excitability by depressing Ca 2+ -influx may be attenuated by down-regulating GABA A receptors surface expression. Together, our results demonstrated that baclofen-induced inhibition of CX43 and CX36 surface expression might promote cytoprotective autophagy by improving GABA A receptor α 1 subunit surface expression.
Furthermore, mitophagy plays a protective effect during cerebral ischemia 85,86 . A significant increase in CX43 and CX36 mitochondrial expression during chronic cerebral hypoperfusion might inhibit mitophagy, impede mitophagy-related mitochondrial clearance, and then aggravate ischemia-induced neuronal cell death. Baclofen-induced inhibition of CX43 and CX36 mitochondrial expression might promote mitophagy, and then inhibit downstream apoptosis. Further investigations are underway in our laboratory.
In conclusion, our present results demonstrated that, under chronic cerebral hypoperfusion, activation of GABA B receptors suppressed cytodestructive autophagic activity through Akt/ERK-Bcl2-Beclin1 signaling pathway, while up-regulated protective autophagy through the activation of GABA A receptor-CX43/ CX36 signaling pathway. The bi-directional regulative effects of GABA B receptors activation on autophagy reversed neuronal damage induced by 2VO (Fig. 10). College, Huazhong University of Science and Technology. Animals were group-housed with free access to water and food with a 12 h light/dark cycle and a thermoregulated environment, and adapted to these conditions for at least 7 days before experiments. All efforts were made to minimize both the suffering and to reduce number of animals used.
OGD-Rep injury of brain slices and drug treatment. The rats were anesthetized with chloral hydrate (350 mg/kg, intraperitoneal injection, i.p.). The cerebrum was removed rapidly and placed in ice-cold artificial cerebrospinal fluid (aCSF) bubbled with 95% O 2 and 5% CO 2 . The aCSF containing (mM): NaCl 126, KCl 3.5, NaH 2 PO 4 1.2, mgCl 2 1.3, CaCl 2 2.0, D-(+ )-glucose 11, NaHCO 3 25; 290 mosm, gassed with 95% O 2 and 5% CO 2 (pH7.4). The cerebrum was immediately sectioned with a Mcllwain tissue chopper (The Mickle Laboratory Engineering Co. LTD, USA) into 400 μ m coronal slices in ice-cold aCSF bubbled with 95% O 2 and 5% CO 2 . The slices were placed on top of the semiporous membranes in six well trays. All the slices were maintained in the plating medium contained DMEM-F/12, 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, US), penicillin and streptomycin (100 U/ml) at 37 °C for 30 min recovery. For OGD, the medium was replaced with sugar-free DMEM, gassed with 95% N 2 and 5% CO 2 at 37 °C for 30 min. For reperfusion, the slices were refreshed with normal culture medium for 6 h. Baclofen (100 μ M) 37 was dissolved in normal culture medium and added to the slices at the onset of reperfusion. Animal model of chronic cerebral hypoperfusion and treatment schedules. Model preparation was described in detail in our previous study 87 . Two weeks after chronic cerebral hypoperfusion, repeated drug treatment of all groups was performed once daily at 20:00 p.m.-21:00 p.m. during the last 21 days. Baclofen (Meryer Chemical Technology Co., Ltd, Shanghai, China) was dissolved in saline at concentration of 1.25 mg/ml and 2.5 mg/ml 31,35,36 . All groups were treated with baclofen in a volume of 10 ml/kg or same volume of normal saline (NS) by i.p. injection. Five weeks after 2VO, rats were killed by decapitation under anesthesia and carried out biochemical studies as described below.
Immunofluorescence and Hematoxylin and Eosin (H&E) staining. After the successive perfusion of rats by intracardiac injection of 0.9% saline solution and 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), brains were harvested and postfixed in 4% PFA overnight. After conventional paraffin embedding and serial section (5 μ m), immunohistochemistry staining was sequentially performed following incubation in anti-LC3 (1: 500, PM036, MBL) overnight at 4 °C and Fluorescein (FITC)-conjugated Affinipure Donkey Anti-Rabbit IgG(H+ L) (SA00003-8, Proteintech Group Inc, China) for 2 h, and imaged with Olympus FluoView 1200 confocal microscope system (Olympus Corporation, Japan). A quantitative analysis of LC3 staining was performed using analySIS software (analySIS 3.0; Soft Imaging System) 88 . TUNEL assay uses the In Situ Cell Death Detection Kit (11684817910, Roche, Basel, Switzerland) according to the manufacturer's protocol. Briefly, the paraffin sections were dewaxed and rehydrated, followed by incubation with protease K (20 mg/ml) for 30 min at 37 °C and the TUNEL reaction mixture 2 h at 37 °C. The nuclei were counterstained with DAPI. Numbers of total nuclei and TUNEL-positive nuclei were counted and the apoptosis ratio was calculated as follows: apoptosis ratio = (number of TUNEL-positive nuclei/number of total nuclei) × 100%. Hematoxylin and eosin staining was performed as follows: hematoxylin staining for 15 min, hydrochloric acid alcohol solution for 35 s decoloring, eosin staining for 10 minutes and 90% ethanol for 40 s decoloring. Then neutral balsam was used for mounting and the section was observed and photographed under the microscope.
Statistical Analysis. All analyses were performed using SPSS 16.0 software (SPSS Inc., USA) and data were presented as mean ± SD. Differences between groups were evaluated using one-or two -way analysis of variance (ANOVA), as appropriate. The t-test was used for testing differences between two groups. P < 0.05 was considered statistically significant.