Background
Histone deacetylase inhibitors (HDACis) are a heterogeneous group of agents that inhibit histone deacetylases (HDACs) and promote posttranslational acetylation of lysine residues within nuclear and cytoplasmic proteins, which may alter their activity and function. In particular, HDAC inhibition can have a profound effect on the acetylation status of histone proteins within chromatin, resulting in the augmented expression of genes relevant to protection from an ischemic insult. In addition, inhibition of deacetylation equally promotes the acetylation of non-histone proteins, such as transcription factors, signal transduction mediators, determining their interaction, localization, and stability [
1]. It is very likely that the non-specificity of deacetylase inhibitors is responsible for the opposing effect noted in distinct type of cells. As it is becoming apparent, HDAC inhibition promotes the demise of tumor cells. The same drugs display strong protective properties for neurons in in vitro and in vivo models of neurotoxicity and neurodegeneration (for rev see [
2]).
Furthermore, it was reported recently that the treatment of adult animals with histone deacetylase inhibitors, such as trichostatin A (TSA), sodium butyrate (SB), and vorinostat (SAHA), administered just before as well as after the onset of stroke, provides neuroprotection [
3‐
8]. The neuroprotective effect of these agents has been associated with decreasing the lesion volume, neurobehavioral improvement, and stimulation of neurogenesis in the ischemic adult brain [
9,
10]. Despite the growing number of evidence supporting the beneficial effect of HDACis in the experimental model of stroke in adult rodents, only a few available reports were addressed upon their effect in the hypoxia-ischemia (HI)-injured immature brain [
11‐
13]. However, due to different experimental paradigms, it is not possible to make the explicit conclusion.
Neonatal HI encephalopathy still remains one of the most important causes of neonatal mortality and/or long-term neurological sequelae such as cerebral palsy, seizure disorders, cognitive and intellectual deficits, and behavioral problems [
14‐
17]. Currently, there are no well-established treatments to reduce brain damage and it is still a big challenge to protect the newborns’ brain from HI injury. The only available effective treatment, hypothermia, neither provides complete brain protection nor stimulates the repair necessary for neurodevelopmental outcome. Recently, HDACis are being considered as valuable tools to reduce or even to prevent HI-induced brain damage in neonates. Since many aspects of the evolving brain damage following the insult differ between adults and neonates, extrapolating data obtained in the mature brain to neonates is generally unwise. Therefore, the present study was undertaken to examine whether treatment with one of the HDACis, sodium butyrate (SB), has neuroprotective effects in a rat model of neonatal HI. We aimed to assess whether SB action is associated with changes in molecular mediators that are crucial for inducing cerebral damage and thus be targeted for therapy. As inflammation is a well-recognized pathogenic factor in perinatal brain injury, we analyzed the microglial and astrocytic cell response to SB treatment and the influence of SB on cytokines, transcription factors, HSP70, and pro- and anti-apoptotic proteins.
Methods
Experimental animal work was conducted according to regulations following European Union directives. Experimental procedures were approved by the Local Ethics Committee for Animal Experimentation. All efforts were made to minimize the number of animals and animal suffering in every step.
Experimental neonatal hypoxia-ischemia
Animals were housed under controlled temperature (22 °C ± 2), with a 12-h light cycle period and pelleted food and water ad libitum. Cerebral hypoxia-ischemia was produced in 7-day-old (P7) Wistar rats of either sex by a permanent unilateral common carotid artery ligation, followed by systemic hypoxia [
18,
19]. As was previously reported, the ligation alone does not decrease cerebral perfusion below critical levels and the addition of hypoxia is required to cause brain infarct [
20]. Briefly, pups were anesthetized with isoflurane (4% induction, 2% maintenance) carried by O
2. Once they were fully anesthetized, a midline neck incision was made and the left common carotid artery was exposed, double ligated with surgical silk, and cut between two ligatures. The incision was then sutured with monofilament nylon. Sham-operated animals underwent the same surgical procedure without the ligation of the carotid artery. The time length of anesthesia lasted on average 5 min. After surgery, the rat pups were returned to their home cage for 1 h to recover. Later, the animals were placed for 1 h in a hypoxic chamber containing 7.6% oxygen balanced with nitrogen with controlled humidity and temperature maintained at 35 °C.
The undamaged hypoxic hemisphere, as well as age-matched sham-operated animals, served as controls. Pups from each litter were randomly assigned to four experimental groups (5 rats per group): (1) control group (vehicle treatment), (2) control animals (SB treatment), (3) animals which underwent HI (vehicle treatment), and (4) animals which underwent HI (SB treatment). Animals were sacrificed at specific time points (12, 24, 48, 72 h and 6 days) after the injury.
Drug administration
Rats subjected to HI or sham operated were treated once a day with subcutaneous injections of sodium butyrate (SB; Sigma-Aldrich; 300 mg/kg body wt) [
4] or vehicle (saline) starting immediately after hypoxic exposure and lasting up to 5 consecutive days.
Tissue preparation
Six days after HI-anesthetized animals were perfused transcardially first with phosphate-buffered saline (PBS) followed by a fixative solution (4% paraformaldehyde, PFA, in 0.1 M phosphate buffer, pH 7.4). The brains were removed and submerged in the same fixative solution for 4 h at 4 °C. Following postfixation brains were cryoprotected overnight in 30% sucrose solution (in 0.1 M PBS), frozen rapidly using dry ice, and placed in −80 °C storage.
For biochemical analysis, animals were sacrificed (12, 24, 48, 72h and 6 days after HI) through decapitation and the whole hemispheres were frozen on dry ice. All tissue samples were stored at −80 °C until used.
Brain injury evaluation
Hematoxylin-eosin (HE) staining was performed to evaluate the neuroprotective effect of SB against ischemia-induced brain damage. Six days after the insult (at postnatal day 13), the pups were anesthetized with 100 mg/kg ketamine combined with 10 mg/kg xylazine and perfused. The brains were dissected and frozen on dry ice. Coronal cryostat sections (20 μm) were stained with HE and examined using light microscopy.
Immunohistochemistry
The following antibodies (source and final dilution) were used for tissue staining: mouse monoclonal anti-ED1 (CD68) (AbD Serotec, 1:100), goat polyclonal anti-Arg-1 (arginase-1) (Santa Cruz, 1:250), rabbit polyclonal anti-IL-1β (Santa Cruz, 1:250), rabbit polyclonal anti-GFAP (Glial Fibrillary Acidic Protein, DAKO, 1:200), and chicken polyclonal anti-GFAP (Millipore, 1:200).
Coronal cryostat sections of the brain (30 μm thick) were cut at the level of the lateral ventricles in serial order to create 10 series sections. Double fluorescent immunohistochemistry was performed on free-floating sections. After blocking for unspecific reactivity, adjacent series of sections were stained for a specific cell-lineage marker.
For identification of the type of microglia, we used markers labeling M1 (ED1/IL-1β) and M2 (ED1/arginase-1) cells. Double labeling was also employed for monitoring astrocytes expressing IL-1β. Tissue sections were rinsed in PBS and then incubated in 10% normal goat serum in PBS containing 0.25% Triton X-100 for 60 min in room temperature (RT). Next, the sections were washed with PBS and incubated with anti-ED-1 or anti-GFAP overnight at 4 °C. The following day, tissue sections underwent the washing procedure, and the primary antibodies were revealed by applying appropriate secondary FITC-conjugated antibodies (AlexaFluor, 1:500) for 60 min at room temperature and in the dark. After this step, the sections were rinsed in PBS and incubated with primary antibodies (anti-Arg-1 or anti-IL-1β) overnight at 4 °C. The next day, after being rinsed in PBS, the sections were exposed to appropriate Cy3-conjugated secondary antibodies (AlexaFluor, 1:500) for 1 h at room temperature. Nuclei were subsequently labeled with the fluorescent dye Hoechst 33258 (2 μg/ml PBS; Sigma).
Labeling was verified using a confocal laser scanning microscope (LSM 780, Carl Zeiss, Germany) using a 10× or 20× objective. A helium-neon laser (543 nm) was utilized in the excitation of Alexa Fluor 546, while an argon laser (488 nm) was applied in the excitation of FITC. ImageJ software was used for quantitative analysis of immunoreactive sections. Five animals per group were analyzed. Images from five sections per animal were taken, and the number of positive-labeled cells as well as fluorescence intensity was assessed in an area of 1.44 mm2.
Determination of cytokine expression in brain extracts
Concentrations of chemokines/cytokines were measured in extracts from brain hemispheres using the EMD Millipore’s MILLIPLEX® MAP Rat Cytokine/Chemokine Magnetic Bead assay according to the manufacturer’s instructions. The cytokines and chemokines analyzed included TNFα, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-12, IFN-γ, and chemokine CXCL10 (IP-10). The median fluorescence intensity plates were assayed on a Bio-Plex® 200 Luminex system with Bio-Plex Manager 5.0 software. The five-parameter logistic method was applied to estimate cytokine/chemokine concentrations in brain homogenates.
Quantitative polymerase chain reaction (real-time PCR)
Gene expression of pro-inflammatory (TNFα, IL-1β) cytokines was evaluated in the brain hemispheres obtained from rats 12, 48 and 72 h after HI. Total RNA was isolated with TRIzol Reagent, and the quality and concentration of RNA was verified by spectrophotometry with the Nanodrop™ apparatus. The samples containing 1 μg of total RNA were reverse transcripted using High Capacity RNA-to-cDNA Kit (Applied Biosystems) according to the manufacturer’s instructions.
Quantitative real-time PCR analyses of cDNA samples (300 ng) with designed specific primers (Table
1) and Fast SYBR Green Master Mix (Applied Biosystems) were performed in 7500 Fast Real-Time PCR System (Applied Biosystem). Reaction parameters were as follows: (1) holding stage, 20 s at 95 °C; (2) cycling stage (40×), 3 s at 95 °C and 30 s at 60 °C; and (3) melt curve stage, 15 s at 95 °C, 1 min at 60 °C, 15 s at 95 °C, and 15 s at 60 °C. Each sample was tested in triplicate during two analyzing sessions. The fluorescence signal from specific transcript was normalized against that of reference gene (SDHA), and threshold cycle values (ΔCt) were quantified as fold changes by the 2
−ΔΔCT method.
Table 1
List of designed primers used in reverse transcription and quantitative real-time (RT)-PCR analysis
IL-1β | 5′-CACCTCTCAAGCAGAGCACAG-3′ | 5′-GGGTTGCATGGTGAAGTCAAC-3′ |
TNFα | 5′-AAATGGGCTCCCTCTCATCAGTCC-3′ | 5′-TCTGCTTGGTGGTTTGCTACGAC-3′ |
SDHA | 5′-CCCTGAGCATTGCAGAATC-3′ | 5′-CATTTGCCTTAATCGGAGGA-3′ |
Western blot analysis
The following antibodies (source and final dilution) were used for analysis: mouse monoclonal anti-NFκB (Cell Signaling, 1:1000), rabbit polyclonal anti-p53 (Cell Signaling, 1:1000), rabbit polyclonal anti-HSP70 (Cell Signaling, 1:1000), rabbit monoclonal anti-COX-2 (Cell Signaling, 1:1000), rabbit polyclonal anti-Bcl-2 (Cell Signaling, 1:1000), rabbit polyclonal anti-Bax (Cell Signaling, 1:1000), and mouse monoclonal anti-actin (MP Biomedicals, 1:500).
Brain tissues were homogenized in RIPA lysis buffer (10 mM Tris-HCl pH 7.5 containing 150 mM NaCl, 1% Nonidet P40, 0.1% SDS, 1% Triton X-100, PMSF 0.1 mg/ml) and a proteinase and phosphatase inhibitor cocktail (Life Technologies, 1:100). Lysates were clarified by centrifugation at 13000 g for 10 min at 4 °C. The supernatant was collected, and protein concentrations were determined using a Bio-Rad DCTM protein assay kit (Bio-Rad). Samples (50 μg protein) were ran on 10–15% SDS-PAGE gels and transferred onto nitrocellulose membranes (Amersham Bioscience). After blocking, membranes were probed with specific primary antibodies and then incubated with horseradish peroxidase-conjugated secondary IgG antibodies (Sigma-Aldrich). Immunoblot signals were visualized using ECL chemiluminescence kit (GE Healthcare Life Sciences). To verify an equal loading of protein per line, the β-actin antibody was used as an internal control for each immunoblotting. Semi-quantitative evaluation of protein levels detected by immunoblotting was performed by computer-assisted densitometric scanning (LKB Utrascan XL, Program GelScan). The level of protein immunoreactivity was determined by frequent analysis of multiple immunoblots.
Quantitative measurement of prostaglandin E2 protein concentration
To estimate the amount of prostaglandin E2 (PGE2) in homogenates obtained from the brain hemispheres, the Prostaglandin E2 ELISA Kit-Monoclonal (Cayman) test was applied according to the supplier’s instructions. Frozen hemispheres were homogenized in 1 ml of 0.1 M phosphate buffer (pH 7.4) containing 1 mM EDTA and 10 μM indomethacin. Homogenates were clarified by centrifugation at 8000 g for 10 min at 4 °C, and the supernatant was collected for analysis. Protein concentrations were determined using a Bio-Rad DC™ protein assay kit (Bio-Rad). After performing the Sandwich ELISA assay, the plates were read at 412 nm using a spectrophotometric plate reader Fluorostar Omega (BMG LabTech).
Quantitative measurement of caspase-3 activity
To estimate the level of activated caspase-3 in lysates obtained from both brain hemispheres, the Caspase-3 Fluorescence Assay Kit (Cayman Chemical) was applied according to the supplier’s instructions. Briefly, the kit employs a specific caspase-3 substrate, N-Ac-DEVD-N'-MC-R110, which, upon cleavage by active caspase-3, generates a highly fluorescent product that is easily quantified. The fluorescence intensity of each was well read using a spectrophotometric plate reader Fluorostar Omega (BMG LabTech; excitation = 485 nm, emission = 535 nm).
Statistical analysis
GraphPad PRISM 5.0 software was used for the statistical analysis of the received data. Comparisons between animal groups were performed using the one-way analysis of variance (ANOVA) followed by the Bonferroni post-hoc test for multiple comparisons or Student’s t test. All values are expressed as mean ± SD. The data were considered significant at p value <0.05.
Discussion
The principal finding in our present study is that sodium butyrate treatment exhibits brain-protective activity in a neonatal hypoxia-ischemia model. The protection afforded by SB was expressed by a clear reduction of brain damage, suppression of brain edema, and preservation of brain architecture when analyzed at 6 days after the onset of hypoxia-ischemia. Furthermore, the effect of SB was associated with substantial inhibition of HI-induced inflammation. Our findings remain in general agreement with those reported previously that deacetylase inhibitors (VPA, TSA, SB) are neuroprotective in cerebral injury models in adult rodents [
3‐
5]. Our data also agrees with a brief paper showing neuroprotection following treatment with valproate (VPA) after HI in neonatal rat [
12].
Neonatal hypoxia-ischemia triggers a series of pathophysiological processes (including loss of energy, acidosis, excitotoxicity, elevation of intracellular calcium, induction of oxidative stress, inflammation) that result in a loss of neurons and severe neurological deficits. It is generally accepted that one of the most important pathogenic components of neonatal brain damage is inflammation induced by either the production of cytokines and chemokines followed by leukocyte (including monocytes and macrophages) infiltration or glial activation and proliferation [
21‐
23]. First of all, it is in agreement that blocking the inflammatory reaction promotes neuroprotection and, in addition, has potential for use in the clinical treatment of ischemic brain injury [
21,
24,
25].
Convincing evidence reveals that HDACis, among VPA, TSA and SB, are efficacious neuroprotective agents in adult cerebral injury models associated with inflammation. Administration of these compounds after the onset of stroke results in a marked reduction of microglia number, suppression of their activation, and inhibition of other inflammatory markers, which in turn lead to improved neuropathological outcome [
4,
5]. In contrast to these findings, our results show that SB treatment of neonatal HI induced a paradoxical significant increase in the number of ED1-positive cells (microglia/macrophages) in the damaged ipsilateral hemisphere at 6 days after the insult, as compared to animals treated with vehicle. As demonstrated in the current study, the majority of ED-1+ cells present a positive reaction with an established marker of M2 microglia phenotype, arginase-1, mostly pronounced in the SB-treated rats. It may be speculated that SB facilitates conversion of M1 to M2 leading to anti-inflammatory signalling and, by this, keeps microglia from acquiring a proinflammatory phenotype, and in consequence prevents tissue damage, such as that found in models of AD, MS, and neurodegeneration [
26‐
28]. This prediction may be reinforced by the parallel decrease in the number of ED-1/IL-1β positive cells observed in our study. The reduced cytokine response after SB treatment, despite an increase in the number of microglia, implies that these cells are not necessarily damaging and in some conditions may alleviate harmful consequences of injury. This hypothesis remains in line with data showing that transition in the microglial response during recovery from the proinflammatory (M1) to immunomodulatory and neurotrophic response (M2) [
29‐
32] and then maintenance of endogenous neurogenesis [
33‐
36] may play a key role in attenuation of brain damage [
37]. To confirm the role of the microglial reaction to HI injury in the developing brain and, in particular, to define the time course of M1 to M2 polarization, further studies will be needed.
It is commonly known that reactive astrocytosis also appears to be a part of the hypoxia-ischemia-induced pathological processes [
38,
39]. Consistent with previous reports [
30,
40,
41], we noted a delayed increase in GFAP expression accompanied with hypertrophy and cell proliferation in the ipsilateral hemisphere at 6 days after the insult, implying astrogliosis. The expression of GFAP was further markedly increased by SB treatment. However, this increase was associated with the reduction of cell population co-expressing GFAP and proinflammatory IL-1β. It is worthy to note that SB treatment also led to diminished IL-β production in microglia/macrophages at the same time point. This reduction of IL-1β expression after SB injection in glial cells parallels the attenuation of brain damage. The precise molecular mechanism responsible for the effect of SB is not known. However, apart from the number of biochemical and morphological factors functioning in concert to influence the final SB effect, accumulation of GFAP protein presented here is likely to also contribute to neuroprotection after neonatal HI. This may be supported by data showing that GFAP knock-out mice have a greater susceptibility to ischemic injury [
42]. Furthermore, experimental disruption of astroglial scar formation following stroke results in an increased spread of inflammation and increased lesion volume [
43]. Although results obtained from adult experiments cannot be directly transferred and used as explanation for neonatal data due to differences in the level of maturation and different ischemia model, some hypotheses may be valid in adults as well as in neonates. Nevertheless, a precise role of enhanced astrogliosis seen after SB treatment of neonatal HI is yet to be determined.
Cytokines are regarded as pro- or anti-inflammatory, and based on their state and/or concentration, they can be protective or harmful. Although these proteins can be found in almost any nucleated cell within the brain, such as brain endothelial cells or neurons, they are mainly produced by glial cells or by immune cells, such as helper T cells. Therefore, in the present study, we followed the influence of SB administration on the total content of selected cytokines correlating with the brain damage. The biological effect of these factors include stimulation and synthesis of other cytokines and prompting leukocyte infiltration, which in turn leads to the induction of neuronal injury mediators and influencing glial expression (see rev [
44]). Our results, in general accordance with other reports [
40], depicted a considerable alteration in the expression of IL-1α, IL-1β, TNFα, and chemokine CXCL10 in the ipsilateral hemisphere at 48 h after HI compared to the control one. In line with this, we also observed a significant enhancement in IL-1β and TNFα mRNA level estimated 12 h following the insult. In addition to these early modifications, IL-1β and chemokine CXCL10 protein expression presented a delayed increase after 6 days of recovery suggesting ongoing inflammation. This is in agreement with reported elevation in mRNA and protein level of IL-1β even at 14 days after HI [
45,
46]. Treatment with SB suppressed significantly HI-induced upregulation of chemokine CXCL10 at 48 h and IL-1β at 6 days after HI. In the case of IL-1α and TNFα, the effect of SB was presented only by a non-significant decrease in their level 48 h after the insult, despite a sole, clear reduction in TNFα mRNA expression in the same condition. Probably both factors do not play a prominent role in the protective action mediated by this inhibitor.
The reduction of IL-1β expression presented in our study seems to be particularly important and strongly supported by a number of data showing that downregulation of this cytokine plays a neuroprotective function in the development of HI encephalopathy [
22,
29,
47,
48]. According to research, the decrease of IL-1β production can reverse cell swelling, brain edema, and neurologic function deficiencies induced by HI [
49].
Despite the number of reports focusing on the role of IL-1β, only a few data are available on the potential role of chemokines in the development of HIE [
50]. It was found in a neonatal mouse study of HI injury that mRNA expression of chemokines precedes infiltration of immune cells into the brain, thus proving their relevance in the inflammatory response. It is therefore reasonable to speculate that the reduction of CXCL10 expression observed in the present study participates, at least partially, in the beneficial action of SB. On the other hand, chemokines attract mesenchymal stem cells to home at the lesion site [
51]. Hence, immunomodulatory intervention may have a negative effect upon specific aspects of neurogenesis and thus brain regeneration. Therefore, the question arises if the protective abilities will outweigh the potentially harmful consequences.
It has been suggested that in terms of anti-inflammatory effects, inhibition of COX-2 and subsequent reduction of prostaglandin E2 (PGE2) generation, a major downstream product of COX-2 enzymatic activity, can lead to attenuation of ischemic injury in adult rodents [
52‐
54]. As demonstrated in the current study, SB administration decreased the HI-induced elevated COX-2 expression in the damaged ipsilateral hemisphere. This observation may be related to the reduced level of pro-inflammatory IL-1β at the same time point, as demonstrated by Neeb et al. [
55]. Unexpectedly, the decreased expression of COX-2 after SB treatment seen 6 days post-HI does not result in diminished generation of PGE2. Moreover, the fact that COX-2 and PGE2 levels do not correlate in animal models of induced inflammation is also an interesting finding [
56]. The lack of this correlation suggests that not COX-2 but COX-1 isoform may be expressed and be responsible for maintaining the PGE2 production under brain ischemia [
54,
57‐
59]. Nevertheless, the reason for SB suppression of COX-2 and not PGE2 level in our study is unclear at present and should be explored in the future. Particular attention should be paid to the complexity of enzymatic pathways embedded in PGE2 synthesis and degradation, rather than focusing only on COX-1 and COX-2 concentration. In this context, it is noteworthy that PGE2 under defined conditions may not only contribute to brain damage but rather affect and modulate neuronal function in a positive way through the regulation of microcirculation and synaptic functions [
60,
61].
Several findings indicate that inhibitors of histone deacetylases may also modify diverse targets including, among others, transcription factors such as NFκB and p 53, the HSP family of proteins, and apoptosis-related genes [
4,
62,
63].
A number of reports point to the damaging role of activated by brain ischemia nuclear factor NFκB. This is supported by studies showing that inhibition of NFκB activation after ischemia in adult rodents prevents brain damage in the insulted hemisphere via inhibition of cytokine response [
64‐
67]. However, our findings revealed that following neonatal hypoxia-ischemia, the expression of NFκB increased significantly in both hemispheres, ipsi- and contralateral, despite tissue alterations not being observed in the hypoxic, uninjured side. Moreover, in both hemispheres, the level of NFκB returned to the control value after SB treatment. Thus, the question arises whether the response of NfκB to SB may constitute part of the defense process against HI-induced damage in the ipsilateral side. It is worthy to mention that probably the basal level of NFκB is sufficient for conditions required for neonatal brain development.
An additional suggested factor by which HDACis are reported to mediate neuroprotection in adult cerebral injury models includes HSP70 [
68‐
71]. HSP70 besides functioning as a key member of molecular chaperon system has also been assigned an anti-apoptotic function, although failure to detect protection against apoptosis in neurons overexpressing HSP70 also has been reported [
72]. Nevertheless, most studies describe increased expression of HSP70 as a neuroprotective mechanism in adult rodents after MCAO [
3,
73,
74], as well as after neonatal HI [
75]. The suggested influence of HSP70 action includes inhibition of nuclear transcription factor—NFκB. In contrast to high expression of HSP70 at 12–48 h found by Van den Tweel [
75] in the damaged HI hemisphere, our present results show significant reduction of this protein level at the same time point regardless of exposure to SB. Additionally, the changes in HSP70 expression observed in our studies do not parallel alterations seen in the level of NfκB. The major difference with our study is that we used P7 vs P12 rats and a different time of hypoxia—60 vs 90 min of hypoxia insult used by Van den Tweel [
75]. The reason for the loss of HSP70 may be due to a low rate of its synthesis or increased activity of proteases able to digest HSP70. Also, our results are more clearly in agreement with Sun et al. [
76], showing that HSP70 is only slightly altered, if at all, in P7 neurons after HI. Interestingly, SB treatment caused elevation of HSP70 expression in both brain hemispheres 6 days post-HI. It seems that such delayed response detected in both hemispheres has to be insult independent. It may be also considered that induction of HSP70 after SB treatment may facilitate neuroplasticity during recovery time and improve learning processes [
77].
We also tested if SB-induced neuroprotection in the HI neonatal brain involves changes in the expression of p53-apoptosis regulating transcription factor. The implication that p53 plays a role in the response that follows a hypoxic-ischemic insult stems from the observation that pifithrin alpha, an inhibitor of p53, decreases the number of apoptotic cells in the ischemic brain [
78]. In contrast to the robust upregulation of p53 detected in the adult ischemia model in rodents and inhibition of p53 protein levels by SB [
4], HI induced in neonates with/or without SB treatment did not show any significant effect. Thus, p53 seems to not contribute to the protective effect of SB.
Finally, our results revealed no apparent effect on caspase-3 activation, as well as on expression of anti-apoptotic proteins Bcl-2 and pro-apoptotic Bax. Therefore, these targets probably do not mediate SB-induced neuroprotection.