Characteristics of traumatic brain injury models: from macroscopic blood flow changes to microscopic mitochondrial changes.

Controlled cortical impingement is a widely accepted method to induce traumatic brain injury to establish a traumatic brain injury animal model. A strike depth of 1 mm at a certain speed is recommended for a moderate brain injury and a depth of > 2 mm is used to induce severe brain injury. However, the different effects and underlying mechanisms of these two model types have not been proven. This study investigated the changes in cerebral blood flow, differences in the degree of cortical damage, and differences in motor function under different injury parameters of 1 and 2 mm at injury speeds of 3, 4, and 5 m/s. We also explored the functional changes and mitochondrial damage between the 1 and 2 mm groups in the acute (7 days) and chronic phases (30 days). The results showed that the cerebral blood flow in the injured area of the 1 mm group was significantly increased, and swelling and bulging of brain tissue, increased vascular permeability, and large-scale exudation occurred. In the 2 mm group, the main pathological changes were decreased cerebral blood flow, brain tissue loss, and cerebral vasospasm occlusion in the injured area. Substantial motor and cognitive impairments were found on day 7 after injury in the 2 mm group; at 30 days after injury, the motor function of the 2 mm group mice recovered significantly while cognitive impairment persisted. Transcriptome sequencing showed that compared with the 1 mm group, the 2 mm group expressed more ferroptosis-related genes. Morphological changes of mitochondria in the two groups on days 7 and 30 using transmission electron microscopy revealed that on day 7, the mitochondria in both groups shrank and the vacuoles became larger; on day 30, the mitochondria in the 1 mm group became larger, and the vacuoles in the 2 mm group remained enlarged. By analyzing the proportion of mitochondrial subgroups in different groups, we found that the model mice had different patterns of mitochondrial composition at different time periods, suggesting that the difference in the degree of damage among traumatic brain injury groups may reflect the mitochondrial changes. Taken together, differences in mitochondrial morphology and function between the 1 and 2 mm groups provide a new direction for the accurate classification of traumatic brain injury. Our results provide reliable data support and evaluation methods for promoting the establishment of standard mouse controlled cortical impingement model guidelines.

Extracellular matrix protein laminin ß1 regulates pain sensitivity and anxiodepression-like behaviors in mice.

Patients with neuropathic pain often experience comorbid psychiatric disorders. Cellular plasticity in the anterior cingulate cortex (ACC) is assumed to be a critical interface for pain perception and emotion. However, substantial efforts have thus far been focused on the intracellular mechanisms of plasticity rather than the extracellular alterations that might trigger and facilitate intracellular changes. Laminin, a key element of the extracellular matrix (ECM), consists of one α-, one ß-, and one γ-chain and is implicated in several pathophysiological processes. Here, we showed in mice that laminin ß1 (LAMB1) in the ACC was significantly downregulated upon peripheral neuropathy. Knockdown of LAMB1 in the ACC exacerbated pain sensitivity and induced anxiety and depression. Mechanistic analysis revealed that loss of LAMB1 caused actin dysregulation via interaction with integrin ß1 and the subsequent Src-dependent RhoA/LIMK/cofilin pathway, leading to increased presynaptic transmitter release probability and abnormal postsynaptic spine remodeling, which in turn orchestrated the structural and functional plasticity of pyramidal neurons and eventually resulted in pain hypersensitivity and anxiodepression. This study sheds new light on the functional capability of ECM LAMB1 in modulating pain plasticity and identifies a mechanism that conveys extracellular alterations to intracellular plasticity. Moreover, we identified cingulate LAMB1/integrin ß1 signaling as a promising therapeutic target for the treatment of neuropathic pain and associated anxiodepression.

Midazolam and ropivacaine act synergistically to inhibit bone cancer pain with different mechanisms in rats.

Analgesic strategy of a single drug analgesia in bone cancer pain (BCP) has shifted to combined analgesia with different drugs which have different mechanism. After tumor cell inculation, the activation of signal transducer and activator of transcription (STAT3) and extracellular signal-regulated kinase (ERK) signaling pathway are involved in the development and maintenance of BCP, whereas a decrease in the expression of spinal STAT3 and ERK through using their specific blocker, lead to attenuation of BCP. Hence, in this study, we clarified that intrathecal (i.t.) injection of midazolam (MZL) and ropivacaine (Ropi) induces synergistic analgesia on BCP and is accompanied with different mechanisms of these analgesic effect. Hargreaves heat test was used to detect the analgesic effect of single dose of i.t. MZL, Ropi and their combination on the BCP rats. At consecutive daily administration experiment, thermal hyperalgesia was recorded, and immunohistochemical staining was used to detect the expression of c-Fos, spinal glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecule-1 (IBA-1). Then, western blot analysis was used to examine spinal TSPO, GFAP, IBA-1, pERK/ERK and pSTAT3/STAT3 levels on day 14 after tumor cell inoculation. i.t. MZL or Ropi showed a short-term analgesia dose-dependently, and MZL displayed better effect on inhibition of pSTAT3 expression than pERK, but Ropi was just the reverse, then consecutive daily administrations of their combination acted synergistically to attenuate thermal hyperalgesia with downregulated spinal 'neuron-astrocytic activation' in the BCP rats. i.t. co-delivery of MZL and Ropi shows synergistic analgesia on the BCP with the inhibition of spinal 'neuron-astrocytic activation'. Spinal different signaling pathway inhibition for MZL and Ropi may be involved in this process.

The analgesic effect of rolipram is associated with the inhibition of the activation of the spinal astrocytic JNK/CCL2 pathway in bone cancer pain.

Bone cancer pain (BCP) is one of the most difficult and intractable tasks for pain management, which is associated with spinal 'neuron-astrocytic' activation. The activation of the c-Jun N-terminal kinase (JNK)/chemokine (C-C motif) ligand (CCL2) signaling pathway has been reported to be critical for neuropathic pain. Rolipram (ROL), a selective phosphodiesterase 4 inhibitor, possesses potent anti-inflammatory and anti-nociceptive activities. The present study aimed to investigate whether the intrathecal administration of ROL has an analgesic effect on BCP in rats, and to assess whether the inhibition of spinal JNK/CCL2 pathway and astrocytic activation are involved in the analgesic effects of ROL. The analgesic effects of ROL were evaluated using the Von Frey and Hargreaves tests. Immunofluorescence staining was used to determine the number of c-Fos immunoreactive neurons, and the expression of spinal astrocytes and microglial activation on day 14 after tumor cell inoculation. Enzyme­linked immunosorbent assay (ELISA) was used to detect the expression of pro-inflammatory cytokines [interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α] and chemokines (CCL2), and western blot analysis was then used to examine the spinal phosphodiesterase 4 (PDE4), ionized calcium binding adapter molecule-1 (IBA-1) and JNK levels on day 14 after tumor cell inoculation. The results revealed that ROL exerted a short-term analgesic effect in a dose-dependent manner, and consecutive daily injections of ROL exerted continuous analgesic effects. In addition, spinal 'neuron­astrocytic' activation was suppressed and was associated with the downregulation of spinal IL-1ß, IL-6 and TNF-α expression, and the inhibition of PDE4B and JNK levels in the spine was also observed. In addition, the level of CCL2 was decreased in the rats with BCP. The JNK inhibitor, SP600125, decreased CCL2 expression and attenuated pain behavior. Following co-treatment with ROL and SP600125, no significant increases in thermal hyperalgesia and CCL2 expression were observed compared with the ROL group. Thus, our findings suggest that the analgesic effects of ROL in BCP are mainly mediated through the inhibition of 'neuron­astrocytic' activation, which occurs via the suppression of spinal JNK/CCL2 signaling.

Anxiolytic effect of essential oils of Salvia miltiorrhiza in rats.

This study aims to investigate the anxiolytic effects of essential oil from S. miltiorrhiza in rats. The elevated plus maze test and the social interaction test were performed to evaluate the anxiolytic effects of essential oil. The levels of noradrenaline (NE), dopamine (DA) and serotonin (5-HT) in cerebral cortex of rats as well as the plasma corticosterone (CORT) level were examined in the rats with the treatment of essential oil. The rota-rod test was carried out to exclude any false positive results in experimental procedures related to anxiety disorders. The catalepsy test was carried out to investigate whether essential oil induces the catalepsy. Our results showed that oral administration of essential oil increased the percentage of time spent in the open arms and increased the number of entries to the open arms in the elevated plus maze test. Oral administration of essential oil also increased the time for social interaction in rats. No apparent extrapyramidal symptom (EPS) was observed in the animals with essential oil treatment. The effect of essential oil in the intracellular chloride (Cl(-)) concentration in the cultured human neuroblastoma cells was assessed. Treatment with essential oil (50-100 mg/kg) increased intracellular Cl(-) concentration in the cell culture in a dose-dependent manner, suggesting the involvement of GABAA receptor-Cl(-) ion channel. Together, our data indicate an anxiolytic effect induced by the essential oil from S. miltiorrhiza.

As2O3 induces apoptosis in human hepatocellular carcinoma HepG2 cells through a ROS-mediated mitochondrial pathway and activation of caspases.

Arsenic trioxide (As2O3) has been shown to induce apoptosis in hepatocellular carcinoma cells. However, the molecular mechanism of As2O3-induced apoptosis in the hepatocellular carcinoma cells remains poorly understood. Here, we investigated the impact of As2O3 exposure on the human hepatocellular carcinoma cell line HepG2 and examined the underlying mechanism of cell death. As2O3 induced apoptosis of HepG2 cells in a dose- and time-dependent manner and caused a massive production of reactive oxygen species (ROS). The antioxidant N-acetylcysteine (NAC) was able to prevent As2O3-induced cell death, implying an involvement of ROS in the induction of As2O3-triggered apoptosis. Furthermore, As2O3 initiated apoptosis by triggering of the mitochondria apoptotic pathway as indicated by inhibited Bcl-2 expression, a collapse of the mitochondrial membrane potential (MMP), release of cytochrome c and activation of the caspase cascade. However, these As2O3-induced events can be prevented by NAC. Taken together, these findings suggest that the As2O3 induced apoptosis through a ROS-mediated mitochondrial pathway and activation of caspases.

Prognostic value of p53 alterations in human osteosarcoma: a meta analysis.

Tumor suppressor gene p53 functions as the guardian of the human genome and mutations in p53 contribute to cancer development. However, studies that investigated the potential of p53 as a prognostic marker in osteosarcoma patients have yielded inconclusive results. Based on recommendation of the Cochrane Collaboration, this meta-analysis was conducted using data from the 17 published studies to evaluate the association of p53 alterations with clinical outcome of osteosarcoma patients. Different databases, including MEDLINE, PsycINFO, Scopus, EMBASE, and the Cochrane Central Register of Controlled Trials (CENTRAL) were searched. Prognostic value of p53 alterations was determined by risk ratio (RR). The data showed that p53-positive immunostaining tended to associate with decreased 2-year survival rates (RR, 1.94; 95% CI, 1.43 to 2.64; p < 0.0001, I(2) = 10%). However, the prediction value of RR was smaller with p53 expression than with p53 mutations. Moreover, patients who received neoadjuvant chemotherapy and surgery tended to have a stronger association between p53-positive staining and 2-year mortality compared to the patients treated with surgery only. However, p53-positive staining was not associated with 3-year (RR, 1.64; 95% CI, 0.84 to 3.20; P = 0.15; I(2) = 56%) and 5-year survival (RR, 1.25; 95% CI, 0.78 to 2.01; P = 0.36; I(2) = 70%). The data from the current study suggest that p53-positive osteosarcoma only predicted a decreased short-term survival rate, but not 3- or 5-year survival.

Patchouli alcohol dampens lipopolysaccharide induced mastitis in mice.

Patchouli alcohol (PA), a tricyclic sesquiterpene isolated from Pogostemonis Herba, has been known to exhibit antioxidant, anti-inflammatory, and other important therapeutic activities. The aim of this study was to investigate the effects of PA on LPS-induced mastitis in vivo and the possible mechanism. The mouse model of mastitis was induced by injection of LPS through the duct of mammary gland. Mice were pretreated with dexamethasone or PA 1 h before and 12 h after induction of LPS. The myeloperoxidase activity and inflammatory cytokines production in mammary tissues were determined. The effects of PA on NF-κB signal pathways were analyzed by Western blotting. The results showed that PA inhibited the LPS-induced TNF-α, IL-6, and IL-1ß production in a dose manner. It was also observed that PA attenuated mammary histopathologic changes. Furthermore, Western blot analysis showed that PA could inhibit the phosphorylation of NF-κB and IκB induced by LPS. These results indicate that PA inhibits NF-κB signaling pathways to attenuate inflammatory injury induced by LPS. PA may be a potent therapeutic reagent for the prevention of mastitis.

Birthdate study of GABAergic neurons in the lumbar spinal cord of the glutamic acid decarboxylase 67-green fluorescent protein knock-in mouse.

Despite the abundance of studies on γ-aminobutyric acid (GABA) ergic neuron distribution in the mouse developing spinal cord, no investigation has been devoted so far to their birthdates. In order to determine the spinal neurogenesis of a specific phenotype, the GABAergic neurons in the spinal cord, we injected bromodeoxyuridine (BrdU) at different developmental stages of the glutamic acid decarboxylase (GAD)67-green fluorescent protein (GFP) knock-in mice. We thus used GFP to mark GABAergic neurons and labeled newly born cells with the S-phase marker BrdU at different embryonic stages. Distribution of GABAergic neurons labeled with BrdU was then studied in spinal cord sections of 60-day-old mice. Our birthdating studies revealed that GABAergic neurogenesis was present at embryonic day 10.5 (E10.5). Since then, the generation of GABAergic neurons significantly increased, and reached a peak at E11.5. Two waves for the co-localization of GABA and BrdU in the spinal cord were seen at E11.5 and E13.5 in the present study. The vast majority of GABAergic neurons were generated before E14.5. Thereafter, GABA-positive neuron generation decreased drastically. The present results showed that the birthdates of GABAergic neurons in each lamina were different. The peaks of GABAergic neurogenesis in lamina II were at E11.5 and E13.5, while in lamina I and III, they were at E13.5 and E12.5, respectively. The present results suggest that the birthdates of GABAergic neurons vary in different lamina and follow a specific temporal sequence. This will provide valuable information for future functional studies.

Antioxidant therapy for pain relief in patients with chronic pancreatitis: systematic review and meta-analysis.

BACKGROUND: Currently, there is no specific therapy for chronic pancreatitis (CP). The treatment of micronutrient antioxidant therapy for painful CP has been sporadically used for more than 30 years, however, its efficacy are still poorly understood. OBJECTIVE: The purpose of this meta-analysis is to investigate the safety and efficacy of antioxidant therapy for pain relief in patients with CP. SETTING: University Hospital in China STUDY DESIGN: Systematic review and meta-analysis METHODS: Two authors independently reviewed the search results and extracted data and disagreements were resolved by discussion. Effects were summarized using standardized mean differences (SMDs), weighted mean differences, or odds ratio (OR) according to the suitable effect model. MEDLINE, PsycINFO, Scopus, EMBASE, and the Cochrane Central Register of Controlled Trials  were searched from 1980 through December 2012. Randomized controlled trials (RCTs) that studied antioxidant supplementation for pain relief in patients with CP were analyzed. RESULTS: Nine randomized controlled trials (RCTs) involving 390 patients were included. Overall, there was no association of antioxidant therapy with pain reduction in CP patients (SMD, -0.55; 95% CI, -1.22 to 0.12; P = 0.67). However, antioxidant therapy significantly increased blood levels of antioxidants in CP patients versus the placebo group (SMD, 1.08; 95% CI, 0.74 to 1.43; P < 0.00001). Interestingly, combined antioxidant (selenium, ß-carotene, vitamin C, vitamin E, methionine) therapy was found to be associated with pain relief (SMD, -0.93; 95% CI, -1.72 to -0.14; P = 0.02), while the trials in which a single antioxidant was used revealed no significant pain relief (SMD, -0.12; 95% CI, -1.23 to 0.99; P = 0.83) in CP patients. Strong evidence was obtained that the antioxidants increased adverse effects (OR, 6.09; 95% CI, 2.29 to 16.17, P < 0.01); nevertheless, none was serious. LIMITATIONS: Because of the small sample, a consolidated conclusion cannot be reached based on current RCTs. Large-sample RCTs are needed to clarify the analgesic effect of antioxidants in CP patients. CONCLUSIONS: Combined antioxidant therapy seems to be a safe and effective therapy for pain relief in CP patients. Measures of total antioxidant status may not help to monitor the efficacy of antioxidant therapy for patients with CP.