Screening Serum Biomarkers for Rats Preconditioned with Hyperbaric Oxygen: Potential of Predicting Prognosis for Stroke.

BACKGROUND: Stroke is a major health concern and a leading cause of mortality and morbidity. We and other groups have documented that hyperbaric oxygen preconditioning could significantly alleviate neuronal damage in ischemia‒reperfusion models through various mechanisms. However, we found that some of the subjects did not benefit from preconditioning with hyperbaric oxygen. The preconditioning phenomenon is similar to vaccination, in which the endogenous survival system is activated to fight against further injuries. However, with vaccine inoculations, we could test for specific antibodies against the pathogens to determine if the vaccination was successful. Likewise, this experiment was carried out to explore a biomarker that can reveal the effectiveness of the preconditioning before neuronal injury occurs. METHODS: Middle cerebral artery occlusion (MCAO) was used to induce focal cerebral ischemia-reperfusion injury. 2D-DIGE-MALDI-TOF-MS/MS proteomic technique was employed to screen the differentially expressed proteins in the serum of rats among the control (Con) group (MCAO model without hyperbaric oxygen (HBO) preconditioning), hyperbaric oxygen protective (HBOP) group (in which the infarct volume decreased after HBO preconditioning vs. Con), and hyperbaric oxygen nonprotective (HBOU) group (in which the infarct volume remained the same or even larger after HBO preconditioning vs. Con). Candidate biomarkers were confirmed by western blot and enzyme linked immunosorbent assay (ELISA), and the relationship between the biomarkers and the prognosis of cerebral injury was further validated. RESULTS: Among the 15 differentially expressed protein spots detected in the HBOP group by Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), 3 spots corresponding to 3 different proteins (haptoglobin, serum albumin, and haemopexin) products were identified by MALDI-TOF-MS/MS. Serum albumin and haemopexin were upregulated, and haptoglobin was downregulated in the HBOP group (p < 0.05 vs. Con and HBOU groups). After the western blot study, only the changes in haemopexin were validated and exhibited similar changes in subjects from the HBOP group in accordance with MALDI-TOF-MS/MS proteomic analysis and enzyme linked immunosorbent assay (ELISA) analysis. The serum level of the hemopexin (HPX) at 2 h after HBO preconditioning was correlated with the infarct volume ratio after MCAO. CONCLUSIONS: Haemopexin may be developed as a predictive biomarker that indicated the effectiveness of a preconditioning strategy against cerebral ischaemic injury.

Electroacupuncture pretreatment induces ischemic tolerance by neuronal TREM2-mediated enhancement of autophagic flux.

The mechanism of electroacupuncture (EA) pretreatment-induced neuroprotection remains unclear. In this study, we found that neuronal Triggering receptor expressed on myeloid cells 2 (TREM2) expression was increased and peaked at 48 h and 72 h after ischemia/reperfusion. After specific knockdown of TREM2 in excitatory neurons, neurological function was damaged, and the infarct volume was enlarged. Furthermore, the expression of LC3II/LC3I and Beclin1 was decreased, while the expression of p62 was increased. EA pretreatment enhanced TREM2, LC3II/LC3I and Beclin1 expression while reducing p62 in the ischemic penumbra area. The EA-induced neuroprotective effects and improvements in autophagic flux were abolished by specific knockdown of TREM2 in excitatory neurons. Taken together, our findings provide novel mechanistic insight into EA-induced ischemic tolerance and suggest a promising therapeutic strategy of targeting neuronal TREM2 to treat brain ischemia.

Combined-Acupoint Electroacupuncture Induces Better Analgesia via Activating the Endocannabinoid System in the Spinal Cord.

Electroacupuncture (EA) therapy has been widely reported to alleviate neuropathic pain with few side effects in both clinical practice and animal studies worldwide. However, little is known about the comparison of the therapeutic efficacy among the diverse EA schemes used for neuropathic pain. The present study is aimed at investigating the therapeutic efficacy discrepancy between the single and combined-acupoint EA and to reveal the difference of mechanisms behind them. Electroacupuncture was given at both Zusanli (ST36) and Huantiao (GB30) in the combined group or ST36 alone in the single group. Paw withdrawal mechanical threshold (PWMT) was measured to determine the pain level. Electrophysiology was performed to detect the effects of EA on synaptic transmission in the spinal dorsal horn of the vGlut2-tdTomato mice. Spinal contents of endogenous opioids, endocannabinoids, and their receptors were examined. Inhibitors of CBR (cannabinoid receptor) and opioid receptors were used to study the roles of opioid and endocannabinoid system (ECS) in EA analgesia. We found that combined-acupoint acupuncture provide stronger analgesia than the single group did, and the former inhibited the synaptic transmission at the spinal level to a greater extent than later. Besides, the high-intensity stimulation at ST36 or normal stimulation at two sham acupoints did not mimic the similar efficacy of analgesia in the combined group. Acupuncture stimulation in single and combined groups both activated the endogenous opioid system. The ECS was only activated in the combined group. Naloxone totally blocked the analgesic effect of single-acupoint EA; however, it did not attenuate that of combined-acupoint EA unless coadministered with CBR antagonists. Hence, in the CCI-induced neuropathic pain model, combined-acupoint EA at ST36 and GB30 is more effective in analgesia than the single-acupoint EA at ST36. EA stimulation at GB30 alone neither provided a superior analgesic effect to EA treatment at ST36 nor altered the content of AEA, 2-AG, CB1 receptor, or CB2 receptor compared with the CCI group. Activation of the ECS is the main contributor of the better analgesia by the combined acupoint stimulation than that induced by single acupoint stimulation.

Inhibition of chemokine CX3CL1 in spinal cord mediates the electroacupuncture-induced suppression of inflammatory pain.

PURPOSE: Chemokine CX3CL1 and its receptor CX3CR1 in the lumbar spinal cord play crucial roles in pain processing. Electroacupuncture (EA) is recognized as an alternative therapy in pain treatment due to its efficacy and safety. However, the analgesic mechanism of EA remains unclear. The aim of this study was to investigate whether EA suppressed complete Freund's adjuvant (CFA)-induced pain via modulating CX3CL1-CX3CR1 pathway. MATERIALS AND METHODS: Inflammatory pain was induced by intraplantar injection of CFA to the left hind paw of Sprague-Dawley rats. EA with 2 Hz for 30 mins was given to bilateral Zusanli acupoints (ST36) on the first and third day after CFA injection. Mechanical allodynia and thermal hyperalgesia were tested with von Frey tests and Hargreaves tests, respectively. The expressions of CX3CL1, CX3CR1 and p38 mitogen-activated protein kinase (MAPK) were quantified with Western blots. The release of IL-1ß, IL-6 and TNF-α were evaluated with ELISA. Recombinant CX3CL1 or control IgG were then injected through intrathecal catheters in the EA-treated CFA model rats. The behavioral tests, p38 MAPK activation and cytokine release were then evaluated. RESULTS: EA significantly inhibited inflammatory pain induced by CFA for 3 days. Meanwhile, EA downregulated the expression of CX3CL1 but not CX3CR1 in the lumbar spinal cord of the CFA rats. Besides, activation of p38 MAPK and the release of pain-related cytokines (IL-1ß, IL-6 and TNF-α) were inhibited by EA. Intrathecal injection of CX3CL1 largely reversed the analgesic effect of EA treatment and re-activated p38 MAPK signaling, and resulted in pro-inflammatory cytokines increase in acupuncture-treated rats. CONCLUSION: Our findings indicate that EA alleviates inflammatory pain via modulating CX3CL1 signaling in lumbar spinal cord, revealing a potential mechanism of anti-nociception of EA in inflammatory pain.

Neuroprotective Autophagic Flux Induced by Hyperbaric Oxygen Preconditioning is Mediated by Cystatin C.

We have previously reported that Cystatin C (CysC) is a pivotal mediator in the neuroprotection induced by hyperbaric oxygen (HBO) preconditioning; however, the underlying mechanism and how CysC changes after stroke are not clear. In the present study, we demonstrated that CysC expression was elevated as early as 3 h after reperfusion, and this was further enhanced by HBO preconditioning. Concurrently, LC3-II and Beclin-1, two positive-markers for autophagy induction, exhibited increases similar to CysC, while knockdown of CysC blocked these elevations. As a marker of autophagy inhibition, p62 was downregulated by HBO preconditioning and this was blocked by CysC knockdown. Besides, the beneficial effects of preserving lysosomal membrane integrity and enhancing autolysosome formation induced by HBO preconditioning were abolished in CysC-/- rats. Furthermore, we demonstrated that exogenous CysC reduced the neurological deficits and infarct volume after brain ischemic injury, while 3-methyladenine partially reversed this neuroprotection. In the present study, we showed that CysC is biochemically and morphologically essential for promoting autophagic flux, and highlighted the translational potential of HBO preconditioning and CysC for stroke treatment.

Rosmarinic acid ameliorates PTSD-like symptoms in a rat model and promotes cell proliferation in the hippocampus.

Rosmarinic acid (RA) is an important component of Chinese herbal medicine treatments and has been demonstrated to exert therapeutic effects in mood disorders. The present study was designed to assess the effects of RA on post-traumatic stress disorder (PTSD)-like symptoms, hippocampal cell proliferation and phosphorylation extracellular regulated protein kinases (pERK1/2) expression. We found that administration of RA (10mg/kg) alleviated PTSD-like symptoms in rats exposed to an enhanced single prolonged stress (ESPS) paradigm and restored hippocampal proliferation and pERK1/2 expression. Interestingly, the effects of RA were inhibited by the blockage of the ERK signaling. These data support the use of RA for treating PTSD and indicate that the ERK1/2 signaling cascade may play a critical role in the therapeutic efficacy of RA in treating such conditions.

SirT1 mediates hyperbaric oxygen preconditioning-induced ischemic tolerance in rat brain.

Our previous studies have shown that hyperbaric oxygen preconditioning (HBO-PC) induces tolerance to cerebral ischemia/reperfusion (I/R). This study aimed to investigate whether SirT1, a class III histone deacetylase, is involved in neuroprotection elicited by HBO-PC in animal and cell culture models of ischemia. Rats were subjected to middle cerebral artery occlusion for 120 minutes after HBO-PC (once a day for 5 days). Primary cultured cortical neurons were exposed to 2 hours of HBO-PC after 2 hours of oxygen-glucose deprivation (OGD). We showed that HBO-PC increased SirT1 protein and mRNA expression, promoted neurobehavioral score, reduced infarct volume, and improved morphology at 24 hours and 7 days after cerebral I/R. Neuroprotection of HBO-PC was attenuated by SirT1 inhibitor EX527 and SirT1 knockdown by short interfering RNA (siRNA), whereas it was mimicked by SirT1 activator resveratrol. Furthermore, HBO-PC enhanced SirT1 expression and cell viability and reduced lactate dehydrogenase release 24 hours after OGD/re-oxygenation. The neuroprotective effect of HBO-PC was emulated through upregulating SirT1 and, reversely, attenuated through downregulating SirT1. The modulation of SirT1 was made by adenovirus infection carrying SirT1 or SirT1 siRNA. Besides, SirT1 increased B-cell lymphoma 2 (Bcl-2) expression and decrease cleaved caspase 3. These results indicate that SirT1 mediates HBO-PC-induced tolerance to cerebral I/R through inhibition of apoptosis.