Ischemic post-conditioning facilitates brain recovery after stroke by promoting Akt/mTOR activity in nude rats

Authors

  • Rong Xie,

    1. Department of Neurosurgery, Stanford University School of Medicine, Stanford, California, USA
    2. Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China
    3. Department of Neurosurgery, Shanghai Jingan District Central Hospital, Shanghai, China
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  • Peng Wang,

    1. Department of Neurosurgery, Stanford University School of Medicine, Stanford, California, USA
    2. Department of Neurosurgery, Xuanwu Hospital, Capital Medical University, Beijing, China
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  • Xunming Ji,

    1. Department of Neurosurgery, Xuanwu Hospital, Capital Medical University, Beijing, China
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  • Heng Zhao

    Corresponding author
    1. Department of Neurosurgery, Stanford University School of Medicine, Stanford, California, USA
    2. Stanford Stroke Center, Stanford University School of Medicine, Stanford, California, USA
    • Address correspondence and reprint requests to Heng Zhao, Department of Neurosurgery, Stanford University School of Medicine, MSLS Bldg., Room P306, 1201 Welch Rd., Stanford, CA 94305-5327, USA. E-mail: hzhao@stanford.edu

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Abstract

While pre-conditioning is induced before stroke onset, ischemic post-conditioning (IPostC) is performed after reperfusion, which typically refers to a series of mechanical interruption of blood reperfusion after stroke. IPostC is known to reduce infarction in wild-type animals. We investigated if IPostC protects against brain injury induced by focal ischemia in Tcell–deficient nude rats and to examine its effects on Akt and the mammalian target of rapamycin (mTOR) pathway. Although IPostC reduced infarct size at 2 days post-stroke in wild-type rats, it did not attenuate infarction in nude rats. Despite the unaltered infarct size in nude rats, IPostC increased levels of phosphorylated Akt (p-Akt) and Akt isoforms (Akt1, Akt2, Akt3), and p-mTOR, p-S6K and p-4EBP1 in the mTOR pathway, as well as growth associated Protein 43 (GAP43), both in the peri-infarct area and core, 24 h after stroke. IPostC improved neurological function in nude rats 1–30 days after stroke and reduced the extent of brain damage 30 days after stroke. The mTOR inhibitor rapamycin abolished the long-term protective effects of IPostC. We determined that IPostC did not inhibit acute infarction in nude rats but did provide long-term protection by enhancing Akt and mTOR activity during the acute post-stroke phase.

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Post-conditioning did not attenuate infarction in nude rats measured 2 days post-stroke, but improved neurological function in nude rats and reduced brain damage 30 days after stroke. It resulted in increased-activities of Akt and mTOR, S6K and p-4EBP1. The mTOR inhibitor rapamycin abolished the long-term protective effects of IPostC.

Abbreviations used
CCA

common carotid arteries

IPostC

ischemic post-conditioning

MCA

middle cerebral artery

MCAo

distal MCA occlusion

mTOR

mammalian target of rapamycin

p-Akt

phosphorylated Akt

Ischemic post-conditioning (IPostC) refers to brief reperfusion interruptions that reduce infarction after cerebral ischemia and mitigate neurological deficits (Zhao et al. 2006; Zhao 2009). This contrasts with ischemic pre-conditioning (Zhao et al. 2003) and has demonstrated comparable protective effects (Zhao 2009, 2011). The underlying protective mechanisms of IPostC are associated with its ability to attenuate production of free radicals (Zhao et al. 2006; Zhao 2009), to inhibit apoptotic cell signaling pathways (Xing et al. 2008) and to promote cell-survival signaling pathways (Wang et al. 2008) such as the Akt pathway (Zhao et al. 2006; Gao et al. 2008b; Pignataro et al. 2008; Zhao 2009).

T cells are active in adaptive immunity and play a central role in cell-mediated immunity. Neutrophils and macrophages have been thought to participate in innate immunity and contribute to brain injury induced by stroke but T cells have also been recently shown to have detrimental effects in the ischemic brain (Yilmaz et al. 2006; Hurn et al. 2007; Liesz et al. 2009; Kleinschnitz et al. 2010). T cells, like macrophages and neutrophils, infiltrate the ischemic brain after stroke (Iadecola and Anrather 2011) yet T cell deficits reduce infarct sizes (Hurn et al. 2007). T cell subsets play various roles in stroke. Deficits of either CD4 or CD8 T cells result in smaller infarct sizes (Yilmaz et al. 2006; Kleinschnitz et al. 2010) while deficits of regulatory T cells (Treg) lead to enlarged and delayed infarct sizes (Liesz et al. 2009). Our laboratory recently showed reduced-infarction in Th1-deficient mice and increased infarction in Th2-deficient mice (Gu et al. 2012). Because IPostC protects the ischemic brain by blocking the inflammatory response, and T cells mediate inflammation, we hypothesized that T cells are crucial for the protective effects of IPostC. Speculating that IPostC reduces infarction by blocking T cell function, we predicted that IPostC would not reduce infarct sizes in T cell–deficient nude rats.

As we have reviewed, the protective mechanisms of IPostC include multiple cell-signaling pathways such as the Akt pathway (Zhao 2009, 2011, 2013). IPostC has been shown to promote Akt activity and, conversely, inhibition of Akt blocks the protective effects of IPostC (Gao et al. 2008b; Pignataro et al. 2008). Akt directly and indirectly activates mTOR activity, which then promotes the downstream molecules 4E-BP1 and S6K to enhance cell differentiation, growth, and survival (Martelli et al. 2010; Sabbah et al. 2011). The mTOR pathway's involvement in IPostC has not been reported.

Our study investigated the acute and long-term protective effects of IPostC in T cell–deficient nude rats. We examined the acute effects of IPostC on Akt activity, including Akt phosphorylation and protein levels of the Akt isoforms Akt 1, Akt 2 and Akt 3 as well as phosphorylation of mTOR, 4E-BP1 and S6K. Whether or not mTOR inhibition blocks the protective effects of IPostC was also studied.

Methods

Animal experiments were conducted according to the protocols approved by the Stanford Institutional Animal Care and Use Committee and the NIH Guidelines for Care and Use of Laboratory Animals. Animals were housed under a 12:12 h light/dark cycle with food and water available ad libitum.

Focal cerebral ischemia and post-conditioning

Focal ischemia was induced by 30 min of transient bilateral common carotid arteries (CCAs) occlusion and permanent distal middle cerebral artery (MCA) occlusion in male Sprague–Dawley rats (230–250 g, Charles River Laboratories International, Wilmington, MA, USA) and T cell–deficient rats (230–250 g, RNU rats; Charles River Laboratories International) (Zhao et al. 2006). Rats were anesthetized by 5% isoflurane and maintained by 2–3% isoflurane. Core body temperature was monitored with a rectal probe and kept at 37°C throughout the experiment. A ventral midline incision was made and the two CCAs were isolated. A 2 cm vertical scalp incision was made midway between the left eye and ear. The temporalis muscle was bisected and a 2 mm burr hole was made at the junction of the zygomatic arch and squamous bone. The distal MCA was exposed and cauterized above the rhinal fissure at the intersection of the lateral vein and MCA. The CCAs were occluded for 30 min with suture tightening. IPostC was conducted immediately after reperfusion by 3 cycles of 30 s reperfusions and 10 s occlusions of the bilateral CCAs as described (Zhao et al. 2006). Blood gas, heart rate, respiratory rate and temperature were monitored throughout the surgery and kept within physiological ranges.

General histology and infarct size measurement

Infarction was measured by 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) or cresyl violet staining. Wild-type and nude rats killed at 2 days or 30 days (with or without rapamycin pre-treatment) after stroke were perfused transcardially with cold 0.9% saline followed by 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) (pH 7.4). Brains from wild-type and nude rats killed 2 days after stroke were sectioned into five coronal blocks rostral (level 1) to caudal (level 5) and stained with 1% TTC solution. Brains from nude rats killed 30 days (with or without rapamycin pre-treatment) after stroke were post-fixed in 4% PFA, 20% sucrose for 24 h and sectioned into 30 μm slices and mounted onto glass slides using a cryostat. Slices were stained with cresyl violet. The damaged or lost areas of the infarcted cortex, as indicated by TTC or cresyl violet were measured by a technician blinded to the animal's condition, normalized to the contralateral cortex and expressed as a percentage, as described previously (Zhao et al. 2006; Gao et al. 2008a, b).

Behavioral testing

The home cage, vibrissa-elicited limb use and postural reflex tests were used to quantify motor asymmetry caused by a unilateral cortical stroke and performed by a technician blinded to the experimental conditions (Zhao et al. 2005; Gao et al. 2008b; Ren et al. 2008). Nude rats were divided into four groups: (i) sham surgery without ischemia; (ii) ischemia only for 30 min (control ischemia); (iii) ischemia plus post-conditioning without rapamycin pre-treatment; (iv) ischemia plus post-conditioning with rapamycin pre-treatment. Rats were handled for 3 days before stroke, and baseline tested on the day before surgery. All behavior tests were performed at 1, 2, 3, 7, 14, 21 and 30 days post-stroke by two technicians blinded to the experimental conditions. The methods used in the home cage, vibrissa-elicited limb use, postural reflex and vibrissa-elicited forelimb placement tests were detailed in our previous studies (Zhao et al. 2005; Gao et al. 2008b; Ren et al. 2008).

Protein preparation of in vivo experiments for western blotting

Ischemic nude rat brains corresponding to the peri-infarct area and ischemic core were harvested at 1, 5, 9 and 24 h after stroke onset to investigate the effects of in vivo stroke on protein expression of pAkt, Akt, Akt isoforms, pmTOR, mTOR, pS6K, S6K, p4EBP1 and 4EBP1, including pmTOR and pS6K expression, as well as GAP-43, after rapamycin pre-treatment. The area of peri-infarction refers to the ischemic tissue spared by post-conditioning while the ischemic core refers to the infarcted region in the ischemic brain treated with IPostC, as described (Gao et al. 2008b). Brain tissue from animals that underwent sham surgery without ischemia was also prepared for western blotting. Whole cell protein was extracted from the fresh brain tissue, and a western blot was performed as described with modification (Zhao et al. 2005; Gao et al. 2008b). Briefly, brain tissue was cut into small pieces and homogenized in a glass homogenizer using 7 volumes of the cold cell extraction buffer (Catalog# FNN0001; Invitrogen, Eugene, OR, USA), containing 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (1 : 20, Catalog# P-2714; Sigma, St. Louis, MO, USA). The homogenate was centrifuged at 18 000 g for 20 min at 4°C, and the supernatant was removed for protein detection.

Western blot procedures and antibodies

Twenty micrograms of protein was loaded into each lane and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 4–15% Ready Gel (Catalog #L050505A2; Bio-Rad, Hercules, CA, USA) at 200 V for 45 min. Protein bands were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) at 100 V for 2 h. Membranes were incubated overnight with primary antibodies at 4°C followed by Alexa Fluor 488 donkey anti-rabbit or anti-mouse IgG secondary antibody (1 : 5000, Invitrogen, Eugene, OR, USA) for 1 h in a dark room. Table 1 lists each primary antibody, manufacturer, catalog number and detection method used. Membranes were scanned using Typhoon Trio (GE Healthcare, Amersham, Buckinghamshire, UK). Optical densities of all protein bands were analyzed using IMAGEQUANT 5.2 software (GE Healthcare). Samples from sham surgery were used as controls for experimental samples. All samples were run on the same gel.

Table 1. Antibodies, their concentrations and manufacturers used in the present studies
AntibodiesSourceDilutionsManufacturerCatalog No.Application
  1. IF, immunofluorescence; WB, western blots.

P-Akt (Ser473)Rabbit1 : 1000Cell Signaling (Boston, MA, USA)9271WB
AktRabbit1 : 1000Cell Signaling9272WB
Akt1Rabbit1 : 1000Cell Signaling2938WB
Akt2Rabbit1 : 1000Cell Signaling3063WB
Akt3Rabbit1 : 500Cell Signaling3788WB
P-mTOR (Ser2448)Rabbit1 : 200/1 : 1000Cell Signaling2971IF/WB
mTORRabbit1 : 1000Cell Signaling2983WB
P-S6K p70 (Ser371)Rabbit1 : 500Cell Signaling9208WB
S6K p70Rabbit1 : 500Cell Signaling9202WB
P-4EBP1Rabbit1 : 500Cell Signaling9456WB

4-EBP1

GAP-43

Rabbit

Rabbit

1 : 500

1 : 500

Cell Signaling

Cell Signaling

9452

5307

WB

WB

β-actinMouse1 : 3000SigmaA-5441WB

Drug delivery

To study whether rapamycin, an mTOR inhibitor, abolishes the long-term protection of post-conditioning in nude rats, rapamycin (Calbiochem, Billerica, MA, USA) was dissolved in PBS to a final concentration of 0.1 mM. 10 μL of rapamycin, or vehicle, was infused into the ventricular space ipsilateral to the ischemia, 1 h before ischemia onset, as described (Zhao et al. 2005), at the coordinate point from bregma: anteroposterior, 0.92 mm; mediolateral, 1.5 mm; dorsoventral, 3.5 mm. Infarct size and behavior tests were performed 30 days after stroke as described above.

Immunofluorescence staining and confocal microscopy

For immunofluorescent staining of brain sections, sham nude rats, ischemic nude rats, ischemic nude rats with post-conditioning and ischemic nude rats pre-treated by rapamycin and with post-conditioning were anesthetized and perfused transcardially with 0.9% saline followed by 4°C PFA in PBS (pH 7.4), then fixed with 4% PFA for 24 h. Free floating 30 um sections were cut on a cryostat and stored in anti-freeze solution at −20°C. Sections were incubated in blocking solution containing 5% horse serum (Sigma) and 0.3% Triton X-100 in PBS for 2 h at 20°C followed by incubation in primary antibodies (Table 1) at 4°C overnight. The next day, sections were washed with PBS and incubated for 1 h at 20°C (light shielded) in secondary antibody (Alexa Fluor 647 donkey anti-rabbit or anti-mouse IgG, 1 : 200; Invitrogen) and cover-slipped with 1 drop of Vectashield mounting medium with 4′, 6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Immunofluorescent staining was examined using a Zeiss confocal microscope (Zeiss LSM 510, Thornwood, NY, USA).

Statistical analysis

GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA) was used for statistical analyses. For infarction analyses, one-way anova was used followed by the fisher least significant difference post hoc test. For western blots, two-way anova was used to compare the optical densities of all protein levels of the peri-infarct area versus the core at the stated time points, followed by the fisher least significant difference post hoc test. Two-way anova was used to analyze various protein bands between post-conditioning, post-conditioning with rapamycin pre-treatment and control ischemia. For behavioral tests, one-way repeated measures anova was used to compare a test at different time points in the same group, and two-way anova was used to compare tests between post-conditioning, post-conditioning with rapamycin pre-treatment, control ischemia and sham groups, followed by the fisher least significant difference post hoc test. Tests were considered significant at p-values < 0.05. Data are presented as mean ± SEM.

Results

IPostC reduced infarct sizes in wild-type but not in nude rats measured 2 days post-stroke

As we have reported previously (Zhao et al. 2006; Gao et al. 2008b; Ren et al. 2008), we confirmed in this study that IPostC reduced infarct sizes in wild-type rats measured 2 days post-stroke (Fig. 1). In a result differing from that in wild-type rats, the cortical infarction in nude rats induced by stroke was 27.1 ± 4.3% in control ischemia versus 23.7 ± 4.2% in post-conditioning (n = 8). A statistical analysis predicts that a total of 89 animals are required to reach p < 0.05, thus we conclude that there is no significant difference between the two groups (Fig. 1). In the control group without IPostC, nude rat infarction was smaller than that in wild-type rats (p = 0.023, n = 8).

Figure 1.

Ischemic post-conditioning (IPostC) reduced acute infarct sizes in wild-type rats but not in nude rats. Stroke was induced by transient occlusion of bilateral common carotid arteries (CCA) for 30 min combined with permanent distal middle cerebral artery (MCA) occlusion. Infarct sizes were measured 2 days post-stroke by 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining. (a) Representative TTC staining for infarction and average infarct sizes in wild-type rats, * versus control. (b) Same for nude rats. < 0.05. n = 8/group.

Akt and mTOR activities were enhanced by IPostC

Despite the unaltered infarct size, we hypothesized that IPostC may improve pathological outcomes of neuronal survival at molecular levels. Both peri-infarct region and ischemic core were dissected for western blotting (Fig. 2). Indeed, the results showed that protein levels of p-Akt (phosphorylated Akt) and Akt isoforms (Akt1, Akt2, Akt3) in the Akt pathway were reduced after stroke, and IPostC significantly increased these protein levels at certain time points, in both the ischemic core and peri-infarct region (Fig. 2). Similarly, p-mTOR, p-S6K, p-4EBP1 and total proteins of mTOR, S6K and 4E-BP1 in the mTOR pathway were also reduced by stroke both in the peri-infarct area and in the core after stroke (< 0.05), and IPostC attenuated these reductions (Fig. 3).

Figure 2.

The effects of ischemic post-conditioning (IPostC) on protein levels of p-Akt, Akt and Akt isoforms. Rat brains were harvested at 1, 5, 9, and 24 h post-stroke for western blotting. (a) A diagram shows that peri-infarct area and ischemic core were dissected for western blot. The peri-infarct area (I) is defined as the ischemic region spared by IPostC, whereas the ischemic core (II) is defined as the infarcted ischemic region receiving IPostC. (b) Representative protein bands of pAkt, Akt and beta-actin in rat brains after stroke with and without postconditioning. (c) Representative protein bands of Akt isoforms with bar graphs that represent average protein levels. (d and e) The bar graphs represent average protein levels from ischemic brains.* and ** versus sham, < 0.05 and 0.01, respectively. # is < 0.05, between the two indicated groups. n = 6–8/group.

Figure 3.

The effects of ischemic post-conditioning (IPostC) on protein levels in the mammalian target of rapamycin (mTOR) pathway. (a) Representative protein bands of pmTOR, mTOR and beta-actin. The bar graphs show the statistical results. (b) Representative protein bands and bar graphs for statistical results of pS6K, S6K and beta-actin. (c) Representative protein bands and bar graphs for statistical results of p4EBP1, 4EBP1 and beta-actin. * and ** versus sham, < 0.05 and 0.01, respectively; #, ##, between indicated two groups, < 0.05, 0.01, respectively, n = 6–8/group.

IPostC attenuated neurological deficits and long-term brain injury, which were blocked by mTOR inhibition

As IPostC improved protein expression of the Akt/mTOR cell survival signaling pathways, we tested our hypothesis that IPostC improves neurological function and inhibits the effects of stroke on brain injury up to 30 days post-stroke. The results of cresyl violet staining showed that IPostC reduced brain damage size from 10.2 ± 1.4% to 4.9 ± 1.6% (n = 6–8, < 0.05) measured 30 days post-stroke (Fig. 4).The results of behavioral tests showed that IPostC improved neurological function in nude rats when measured from 1 to 30 days after stroke (n = 6–8, < 0.05). In addition, injection of the mTOR inhibitor rapamycin abolished the long-term protective effects of post-conditioning (Fig. 4).

Figure 4.

Mammalian target of rapamycin (mTOR) inhibition blocked long-term protective effects of post-conditioning on brain injury and behavioral deficits after stroke. (a) Representative Cresyl violet staining for brain injury measured 30 days after stroke. Average infarct sizes are shown in the bar graph. Rapamycin, the mTOR inhibitor, was shown to reverse the protective effects of post-conditioning. * versus con ISC and # versus post + rapa, p < 0.05. (b) Results of vibrissae, home cage and postural reflex tests. Post-conditioning promoted behavioral recovery, which was blocked by rapamycin. * and ** versus sham, p < 0.05 and 0.01, respectively; # versus post, p < 0.05. n = 6–8/group.

Western blot results confirmed that rapamycin injection reduced protein levels of pmTOR and pS6K in brains with or without stroke plus IPostC (Fig. 5a). Confocal microscopy showed that pmTOR expression in the peri-infarct area receiving IPostC was reduced by rapamycin (Fig. 5b).

Figure 5.

Effects of rapamycin on protein levels of pmTOR and pS6K. (a) Western blots show that rapamycin reduced protein levels of pmTOR and pS6K, represented by protein bands of pmTOR and pS6K in both the peri-infarct area and the ischemic core. Animals receiving ischemic post-conditioning (IPostC) were treated with or without rapamycin. The sham group indicates that animals received sham surgery only; the Rapa+Sham group indicates that animals received sham surgery plus rapamycin injection. Rapamycin injection reduced protein levels of both pmTOR and mS6K in the group receiving sham surgery alone or with stroke and IPostC. * and ** versus sham, < 0.05 and 0.01, respectively; & vs rapa + sham, < 0.05; #< 0.05, between indicated two groups, n = 6–8/group. (b) Confocal microscopy shows representative immunofluorescent staining of pmTOR and pS6K. Imaged in a peri-infarct region 24 h post-stroke with IPostC, with and without rapamycin. Scale bar, 60 μM.

As we have found that IPostC did not alter acute infarction but still improved neurological function, we further measured protein levels of pre-synaptic GAP43, which is associated with brain plasticity and neurological function. The results showed that protein levels of GAP43 were immediately decreased after stroke, which were maintained by IPostC treatment. However, rapamycin administration abolished the protective effect of IPostC (Fig. 6).

Figure 6.

The acute effects of rapamycin injection on protein levels of growth associated protein 43 (GAP43) after stroke. (a) Representative protein bands of GAP43. Protein of β-actin was probed to show even loading of proteins for western blotting. (b) Bar graphs show average protein optical densities, which were normalized to sham groups, and expressed as percentages. The protein levels were decreased post-stroke, but ischemic post-conditioning (IPostC) maintained its levels. Rapamycin administration abolished the protective effects of IPostC. *, ** versus sham, p < 0.05, 0.01, respectively; #, between indicated two groups, p < 0.05. n = 6–8/group.

Discussion

We found that IPostC did not reduce infarct sizes measured 2 days post-stroke in nude rats but attenuated neurological deficits and inhibited long-term brain injury measured 30 days post-stroke. Our data imply that T cells are a factor involved in the protective effects of IPostC. Despite unaltered infarct sizes, we showed that IPostC attenuated reductions in pAkt levels, Akt isoform proteins, as well as pmTOR, mTOR, p4E-BP1, and pS6K, as well as GAP43 in the acute ischemic brain. Because rapamycin, an mTOR pathway inhibitor, blocked the long-term protective effects of IPostC on brain injury and neurological deficits, we conclude that IPostC provides protection by promoting mTOR activity in nude rats.

In another set of experiments, we have proven that mTOR also plays a critical role in brain damage after stroke and contributes to the protective effects of IPostC in wild-type rats (unpublished data). As consistent with this study, we found that IPostC attenuated stroke-induced reductions in protein phosphorylation in the mTOR pathway as measured from 1 to 24 h after stroke. Both rapamycin and mTOR shRNA injection worsened infarction and inhibited protection by IPostC. In addition, S6K gene transfer protected against brain injury. Furthermore, we found that mTOR inhibition played a critical role in long-term protection of IPostC, as rapamycin abolished the long-term protection afforded by IPostC on neurological functions and injury size as measured 3 weeks post-stroke. Lastly, we examined if early modulation of mTOR activity had long-term effects on protein levels of p-mTOR, p-S6K and p-4EBP1 in the mTOR pathway. We found that IPostC improved protein levels in the mTOR pathway at least 1 week after stroke, but rapamycin administration resulted in significant reductions in these proteins both at 1 and 3 weeks with IPostC. Taken together, mTOR appears to play similar roles in the protective effects of IPostC in wild-type rats compared with nude rats. Nevertheless, the relationship between T cells and changes in Akt and mTOR pathways after stroke remain elusive. As T cells are involved in inflammation after stroke (Jin et al. 2010; Kleinschnitz et al. 2010), and inflammation may affect Akt and mTOR activity (Guha and Mackman 2002; Weichhart et al. 2011), T cells may affect the Akt and mTOR pathways via inflammatory response. Future studies are required to compare changes in these cell signaling pathways between nude rats and wild-type rats.

In this study, our primary purpose is to address whether IPostC has similar protective effects in nude rats whose T cells are not present, as T cells have been shown to contribute to brain injury (Gu et al. 2012; Xiong et al. 2013). We and others have previously shown that IPostC reduced infarct sizes measured 2 to 3 days after stroke in wild-type rats, and this protective effect lasted 1 month (Zhao et al. 2006; Gao et al. 2008b; Ren et al. 2008). In the T cell–deficient nude rats in our current study, however, IPostC did not reduce infarct sizes measured at 2 days, but reduced brain injury size measured at 30 days post-stroke. These results suggest that T cells are crucial to modulating the acute protective effects of IPostC, and that cell types other than T cells may mediate the long-term protective effects of IPostC.

We and others have previously shown that Akt contributes to the protective effects of IPostC in wild-type rats, as IPostC promotes pAkt levels and PI3K/Akt inhibitors block the protective effects of IPostC (Gao et al. 2008a; Pignataro et al. 2008). Although in this study IPostC improved Akt activity measured 24 h after stroke, this activity apparently did not reduce acute infarct size, but more likely contributed to improved neurological function and long-term outcomes. As the mTOR pathway is downstream of the Akt pathway, we further examined the effects of IPostC on critical proteins in the mTOR pathway. Although some studies have reported on mTOR's involvement in ischemic injury, results regarding mTOR's role in neuroprotection are controversial. For example, inhibition or deletion of PTEN was shown to promote axonal outgrowth or inhibit neuronal death by enhancing mTOR activity (Shi et al. 2011; Mao et al. 2012), and t-PA increased neuronal survival by activating mTOR pathways, resulting in improved energy availability (Wu et al. 2012). mTOR activity was also shown to promote astrocyte survival after cerebral ischemia (Pastor et al. 2009). However, mTOR has also been shown to promote post-ischemic long-term potentiation, which causes increased neuronal death, and mTOR inhibition by rapamycin reduced neuronal damage (Ghiglieri et al. 2010). With regard to the neuroprotective effects of mTOR, we have shown for the first time that IPostC increases protein activity in the mTOR pathway, including pmTOR, p4E-BP1 and pS6K proteins in nude rats. Because injection of the mTOR inhibitor rapamycin abolished the protective effects of IPostC on long-term brain injury size and neurological deficits, we conclude that mTOR is a crucial protein for the long-term protective effects of IPostC.

As IPostC improved neurological function measured 1 day post-stroke, we further hypothesized that it improves functional protein related with neuroprotection. GAP43 is expressed on the pre-synaptic terminals, and is a known growth and plasticity protein (Carmichael 2003). Its over-expression has been associated with improved brain recovery and learning ability following treatment with neuroprotectants after stroke (Yoon et al. 2012). We found that IPostC improved protein levels of GAP43, and rapamycin administration abolished the protective effects of IPostC on GAP43 levels. These results further strengthened why IPostC did not reduce infarction yet improved neurological function.

The detrimental effects of T cells in brain injury induced by stroke have been well documented (Yilmaz et al. 2006; Hurn et al. 2007; Liesz et al. 2009; Kleinschnitz et al. 2010). We recently demonstrated that T cell subsets are responsible for distinct effects in brain injury after stroke. We showed that deficits of CD4 and CD8 T cells resulted in smaller infarct sizes, while the lack of Treg cells had no effect on acute infarction measured at 2 days post-stroke (Gu et al. 2012). We also showed that while Th1 cell deficiency was neuroprotective, Th2 cell deficiency resulted in enlarged infarction (Gu et al. 2012). We recently reported that the detrimental effects of T cells are ischemic model dependent (Xiong et al. 2013). In the distal MCA occlusion (MCAo) model, in contrast to the MCA suture occlusion model, the distal MCA was permanently occluded and the bilateral CCAs were transiently occluded for 60 min (Xiong et al. 2013). This model is similar to the model used in our study, except that the bilateral CCA occlusion was 30 min. In the MCA suture occlusion model the MCA was transiently occluded for 100 min by the insertion of a monofilament suture (Xiong et al. 2013). T cell–deficient nude rats showed an approximate 50% reduction in infarct size in the MCA suture occlusion model when compared to wild-type rats, whereas lack of T-cells had no effect in the distal MCAo model. In our study, however, when the time of bilateral CCAo occlusion was reduced to 30 min T cell–deficient nude rats also had smaller infarctions than wild-type rats. Therefore, the protective effects of T cell deficiency are determined by ischemic models as well as ischemic durations. Our study indicates that T cells inhibition may be responsible for part of the acute protective effects of IPostC because IPostC did not reduce acute infarction in nude rats as in wild-type rats. Future studies should address why and how T cells are involved in IPostC, and why IPostC has long-term protective effects on brain injury.

In summary, T cells appear to have a crucial role in the protective effects of IPostC against stroke-induced brain injury. In the absence of T cells, IPostC did not reduce acute infarction but did improve neurological deficits for at least 30 days, at which time brain injury size was also reduced. It is plausible that this protection is associated with increased Akt and mTOR activity. Our study provides innovative insights into the underlying protective mechanisms of IPostC. Further study is required to address how T cells enhance the protective effects of IPostC and the associated cell-signaling pathways.

Acknowledgements

The authors thank Ms. Cindy H. Samos and Greta Beekhuis for manuscript assistance. This study was supported by AHA (Western State Affiliate) grant in aid 10GRNT4200024 and NIH grant 1R01NS 064136 (HZ). There is no conflict of interest to be disclosed.

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