Sestrin2 induced by hypoxia inducible factor1 alpha protects the blood-brain barrier via inhibiting VEGF after severe hypoxic-ischemic injury in neonatal rats.

OBJECTIVE: Hypoxic ischemic (HI) encephalopathy remains the leading cause of perinatal brain injury resulting in long term disabilities. Stabilization of blood brain barrier (BBB) after HI is an important target, therefore, in this study we aim to determine the role of sestrin2, a stress inducible protein which is elevated after various insults, on BBB stabilization after moderate and severe HI injuries. METHODS: Rat pups underwent common carotid artery ligation followed by either 150min (severe model) or 100min (moderate model) of hypoxia. 1h post HI, rats were intranasally administered with recombinant human sestrin2 (rh-sestrin2) and sacrificed for infarct area, brain water content, righting reflex and geotaxis reflex. Sestrin2 was silenced using siRNA and an activator/inhibitor of hypoxia inducible factor1α (HIF1α) was used to examine their roles on BBB permeability. RESULTS: Rats subjected to severe HI exhibited larger infarct area and higher sestrin2 expression compared to rats in the moderate HI group. rh-sestrin2 attenuated brain infarct and edema, while silencing sestrin2 reversed these protective effects after severe HI. HIF1α induced sestrin2 activation in severe HI but not in moderate HI groups. A HIF1a agonist was shown to increase permeability of the BBB via vascular endothelial growth factor (VEGF) after moderate HI. However, after severe HI, HIF1α activated both VEGF and sestrin2. But HIF1α dependent sestrin2 activation was the predominant pathway after severe HI which inhibited VEGF and attenuated BBB permeability. CONCLUSIONS: rh-sestrin2 attenuated BBB permeability via upregulation of endogenous sestrin2 which was induced by HIF1α after severe HI. However, HIF1α's effects as a prodeath or prosurvival signal were influenced by the severity of HI injury.

Sestrin2 prevents neonatal incision pain and re-incision enhanced hyperalgesia in adult rats.

BACKGROUND: Improving the methods for recognizing pain is important for infants admitted to the neonatal intensive care unit. Sestrin2 is a novel stress-inducible protein with a neuroprotective role that functions as a molecular mediator of hormesis. Nevertheless, the role of sestrin2 in the pain process is still unclear. The following study examined the role of sestrin2 on mechanical hypersensitivity after pups incision, as well as enhanced pain hyperalgesia after adulthood re-incision in rats. METHODS: The experiment was divided into two parts: (1) studying the effect of sestrin2 in the neonatal incision; (2) studying the priming effect in adulthood re-incision. An animal model was established in seven-day-old rat pups with a right hind paw incision. Pups were intrathecally administrated rh-sestrin2 (exogenous sestrin2). Paw withdrawal threshold testing was performed to assay mechanical allodynia; tissue was analyzed in ex vivo using Western blot and immunofluorescence. SB203580 was further used to inhibit microglial function and evaluate the sex-dependent effect in adulthood. RESULTS: Sestrin2 expression increased transitorily in the spinal dorsal horn in pups after incision. Administration of rh-sestrin2 improved pups' mechanical hypersensitivity by regulating the AMPK/ERK pathway and alleviated re-incision-induced enhanced hyperalgesia in male and female adult rats. After administration of SB203580 in pups, the mechanical hyperalgesia following re-incision in adult rats was prevented in males but not females; however, the protective effect of SB203580 in males was counteracted by silencing sestrin2. CONCLUSIONS: These data suggest that sestrin2 prevents neonatal incision pain and re-incision enhanced hyperalgesia in adult rats. Moreover, microglia inhibition affects enhanced hyperalgesia only in adult males, which may be regulated through the sestrin2 mechanism. To sum up, these sestrin2 data may be a potential common molecular target for treating re-incision hyperalgesia in different sexes.

Biphasic activation of extracellular signal-regulated kinase in anterior cingulate cortex distinctly regulates the development of pain-related anxiety and mechanical hypersensitivity in rats after incision.

BACKGROUND: A recent study has demonstrated that surgical incision induces an anxiety-like behavior but its relationship with incision-evoked mechanical hypersensitivity remains elusive. Extracellular signal-regulated kinase (ERK) activity in the anterior cingulate cortex (ACC) is important for the affective pain. The current study aims to explore ERK1/2 activity in the ACC and its role in the development of anxiety and mechanical hypersensitivity after incision. METHODS: Anxiety-like behavior was measured by elevated plus maze experiment and open field test after hind paw incision. ERK1/2 phosphorylation was determined by immunohistochemistry and Western blot. Cannulae were implanted into the bilateral ACC for the intra-ACC injection of ERK inhibitors PD98059 and U0126. Brushing (innocuous stimulus) was used to investigate its effect on ERK activation under the incision-evoked painful condition. RESULTS: The anxiety-like behavior induced by the hind paw incision persisted longer than mechanical hypersensitivity. One hind paw incision resulted in a biphasic ERK activation in bilateral ACC. Inhibiting ERK activation in the early phase attenuated pain-related anxiety and mechanical hypersensitivity whereas inhibiting ERK activation in the late phase only reduced the anxiety-like behavior. During the time interval between two phases of ERK activation, brushing the incised skin dramatically increased ERK phosphorylation in the ACC. CONCLUSIONS: These data suggest that in the early phase of postoperative pain, pain-related anxiety and mechanical hypersensitivity are tightly linked and regulated by the ERK activation in the ACC. However, in the late phase of postoperative pain, ERK activation in the ACC is only required for the expression of pain-related anxiety but not mechanical hypersensitivity.

Sestrin2, as a negative feedback regulator of mTOR, provides neuroprotection by activation AMPK phosphorylation in neonatal hypoxic-ischemic encephalopathy in rat pups.

Hypoxic-ischemic encephalopathy is a condition caused by reduced oxygen and cerebral blood flow to the brain resulting in neurological impairments. Effective therapeutic treatments to ameliorate these disabilities are still lacking. We sought to investigate the role of sestrin2, a highly conserved stress-inducible protein, in a neonatal rat hypoxic-ischemic encephalopathy model. Ten-day-old rat pups underwent right common carotid artery ligation followed by 2.5 h hypoxia. At 1 h post hypoxic-ischemic encephalopathy, rats were intranasally administered with recombinant human sestrin2 and sacrificed for brain infarct area measurement, Fluoro-Jade C, immunofluorescence staining, Western blot, and neurological function testing. rh-sestrin2 reduced brain infarct area, brain atrophy, apoptosis, ventricular area enlargement, and improved neurological function. Western blot showed that sestrin2 expression levels were increased after treatment with rh-sestrin2, and sestrin2 exerts neuroprotective effects via activation of the adenosine monophosphate-activated protein kinase pathway which in turn inhibits mammalian target of rapamycin signaling resulting in the attenuation of apoptosis. In conclusions: Sestrin2 plays an important neuroprotective role after hypoxic-ischemic encephalopathy via adenosine monophosphate-activated protein kinase signaling pathway and serves as a negative feedback regulator of mammalian target of rapamycin. Administration of rh-sestrin2 not only reduced infarct area and brain atrophy, but also significantly improved neurological function.

Sestrins: A New Kid for Stroke Treatment?

BACKGROUND: The sestrin family includes several conserved stress-induced proteins that contribute to the maintenance of homeostasis, DNA stability and cell viability in response to various types of injuries. OBJECTIVE: It is well established that the protective functions of AMP-dependent protein kinase (AMPK) and/or mammalian target of rapamycin (mTOR) are regulated by sestrins. Additionally, it has been revealed that sestrins are able to protect cells from oxidative stress by scavenging reactive oxygen species (ROS). METHOD: The essential involvement of sestrins in mTORC1 inhibition and ROS scavenging signaling pathways, which modulate metabolism homeostasis and regulate autophagy, indicates that sestrins may serve as a potential agent for cell growth, development, metabolism, and neurodegenerative disorders. However, the potential role of sestrins in stroke has not been discussed and summarized. RESULTS & CONCLUSION: Based on the current understanding of sestrins, it is believed that sestrins are one of the potential endogenous protective molecules/mechanisms following cerebral stroke, which are associated with neuronal protection, neuroinflammation suppression, and blood brain barrier preservation.

Management of Spontaneous Subarachnoid Hemorrhage Patients with Negative Initial Digital Subtraction Angiogram Findings: Conservative or Aggressive?

Background. The ideal management of SAH patients with negative initial DSA findings remains unresolved. Objective. (i) To present risk factors, clinical courses, and outcomes in different types of SAH patients with negative DSA findings; (ii) to explore the differences of basal vein between aSAH patients and NASAH patients; and (iii) to evaluate the value of repeated DSA for these patients. Methods. All SAH patients with negative initial DSA findings between 2013 and 2015 in our hospital were enrolled and were further categorized as perimesencephalic SAH (PMN-SAH) or nonperimesencephalic SAH (nPMN-SAH). Risk factors, clinical courses, outcomes, and the basal vein drainage patterns were compared. Results. A total of 137 patients were enrolled in the present study. The PMN-SAH group had better GOS and mRS values at 1-year follow-up. Moreover, the nPMN-SAH group had a higher rate of complications. The basal vein drainage pattern showed significant difference when comparing each of the NASAH subtypes with aSAH groups. There was a significant higher rate of a responsible aneurysm in nPMN-SAH group upon repeated DSA. Conclusions. SAH patients with negative initial DSA findings had benign clinical courses and outcomes. Repeated DSA studies are strongly advised for patients with the nPMN-SAH pattern.

Activation of spinal ERK1/2 contributes to mechanical allodynia in a rat model of postoperative pain.

Extracellular signal­regulated kinase (ERK) 1/2 in the spinal cord has been implicated in the development of neuropathic pain and inflammatory pain. However, a limited number of studies have investigated the role of spinal ERK in incisional pain. The present study aimed to determine the role of ERK in the spinal cord in incisional pain. Incisional pain was established in rats by a unilateral hind paw incision. ERK1/2 expression was analyzed by immunohistochemistry. Hypersensitivity to pain was evaluated by measuring the paw withdrawal threshold using the von Frey test. The mitogen­activated protein kinase kinase (MEK) inhibitor, U0126, was administered 20 min prior to or 10 min following the incision by intrathecal or intraperitoneal injection. Phosphorylated ERK1/2 in the ipsilateral L4­5 spinal superficial dorsal horn was activated 1 min following the incision, reached its peak level at 5 min and then returned to the basal level 20 min following the incision. Pretreatment, but not post­treatment with U0126 markedly attenuated the pain hypersensitivity induced by the incision. Therefore, the present study indicates that the transient activation of spinal ERK1/2 contributes to the initiation of pain hypersensitivity following surgical incision.

Analgesic effects of the COX-2 inhibitor parecoxib on surgical pain through suppression of spinal ERK signaling.

Cyclooxygenase (COX)-2 inhibitors are widely used for postoperative pain control in clinical practice. However, it is unknown whether spinal sensitization is involved in the analgesic effects of COX-2 inhibitors on surgical pain. Extracellular signal-regulated kinase (ERK) in the spinal cord is implicated in various types of pain, including surgical pain. The present study investigated the role of spinal ERK signaling in the analgesic effect of the COX-2 inhibitor parecoxib on surgical pain. Surgical pain was produced in rats by surgical incision of the hind paw. Phosphorylated (p)-ERK1/2 expression was determined by immunohistochemistry. Pain hypersensitivity was evaluated by measuring the paw withdrawal threshold using the von Frey test. The selective COX-2 inhibitor parecoxib was delivered 20 min before or 20 min after the incision by intraperitoneal injection. Pretreatment with parecoxib markedly attenuated the pain hypersensitivity induced by incision. However, post-treatment with parecoxib produced minimal analgesic effects. Parecoxib inhibited the increase in spinal p-ERK expression following surgical incision. The present study thus suggests that the COX-2 inhibitor parecoxib exerts its analgesic effect on surgical pain through the inhibition of neuronal ERK activation in the spinal cord. COX-2 inhibitor delivery prior to surgery has more potent analgesic effects, suggesting the advantage of preventive analgesia for post-operative pain control.

Expression of acetylated histone 3 in the spinal cord and the effect of morphine on inflammatory pain in rats.

In this study, a rat model of inflammatory pain was produced by injecting complete Freund's adjuvant into the hind paw, and the expression of acetylated histone 3 in the spinal cord dorsal horn was examined using immunohistochemical staining. One day following injection, there was a dramatic decrease in acetylated histone 3 expression in spinal cord dorsal horn neurons. However, on day 7, expression recovered in adjuvant-injected rats. While acetylated histone 3 labeling was present in dorsal horn neurons, it was more abundant in astrocytes and microglial cells. The recovery of acetylated histone 3 expression was associated with a shift in expression of the protein from neurons to glial cells. Morphine injection significantly upregulated the expression of acetylated histone 3 in spinal cord dorsal horn neurons and glial cells 1 day after injection, especially in astrocytes, preventing the transient downregulation. Our results indicate that inflammatory pain induces a transient downregulation of acetylated histone 3 in the spinal cord dorsal horn at an early stage following adjuvant injection, and that this effect can be reversed by morphine. Thus, the downregulation of acetylated histone 3 may be involved in the development of inflammatory pain.