Reduction of phosphorylated α-synuclein through downregulation of casein kinase 2α alleviates dopaminergic-neuronal function.

Among the post-translational modifications of α-synuclein, phosphorylation has been reported to modulate the protein's nuclear localization, gene-expression and cytotoxicity. However, its effect on the functional performance of dopaminergic-neurons is not known. We aimed to evaluate the effect of siRNA-silencing of casein kinase (CK)2α in SH-SY5Y-cells overexpressing A53T α-synuclein, in alleviating phosphorylated α-synuclein serine129 (pSyn-129)-induced changes in intracellular Ca2+ ([Ca2+]i) response to physiological stimuli and vesicular-dopamine release. A53T transfection showed distinct increase in basal pSyn-129 expression with simultaneous nuclear localization, and CK2α siRNA decreased ROS-generation and pSyn-129 levels. A significant reduction was observed in KCl-induced ([Ca2+]i) response and vesicular-dopamine release in the A53T-transfected cells with a corresponding decrease in immunopositive-population of resting-vesicles (VMAT2). CK2α siRNA treatment showed recovery in [Ca2+]i rise with a corresponding upregulation of expression of voltage-gated Ca2+-channels (VGCC) CaV1.3 and CaV2.2 and RyR1 responsible for Ca2+ induced Ca2+ release from ER, VMAT2 expression and vesicular-dopamine release. Thus, using CK2α siRNA to reduce phosphorylation improved cellular-pathology in terms of ROS generation and pSyn-129 levels, as well as functional performance of DA-neuronal cells.

Impact of monomeric and aggregated wild-type and A30P/A53T double-mutant α-synuclein on antioxidant mechanism and glutamate metabolic profile of cultured astrocytes.

Serving as a source of glutathione and up-taking and metabolizing glutamate are the primary supportive role of astrocytes for the adjacent neurons. Despite the clear physical association between astrocytes and α-synuclein, the effect of extracellular α-synuclein on these astrocytic functions has not yet been elucidated. Hence, we aim to assess the effect of various forms of α-synuclein on antioxidant mechanism and glutamate metabolism. Wild-type and A53T/A30P double-mutant α-synuclein, both in monomeric and aggregated forms, were added extracellularly to media of midbrain rat astrocyte culture, with their survival, oxidative, and nitrative stress, glutathione and glutamate content, expression of enzymes associated with oxidative stress and glutamate metabolism, glutamate and glutathione transporters being assessed along with the association/engulfment of these peptides by astrocytes. A30P/A53T peptide associated more with astrocytes, and low-extracellular K+ concentration showed prominent reduction in the engulfment of the monomeric forms, suggesting that the association of the aggregated forms was greater with the membrane. The peptide-associated astrocytes showed lower survival and increased oxidative stress generation, owing to the decrease in nuclear localization of Nrf2 and increase in iNOS, and further aggravated by the decrease in glutathione content and related enzymes like glutathione synthetase, glutathione peroxidase, and glutathione reductase. Glutamate uptake increased in aggregate-treated cells due to the increase in GLAST1 expression, de novo synthesis of glutamate by pyruvate carboxylase, and/or glutamine synthase, bolstered by the differential glutamate dehydrogenase enzyme activity. We thus show for the first time that extracellular α-synuclein exposure leads to astrocytic dysfunction with respect to the antioxidant mechanism and glutamate metabolic profile. The impact was higher in the case of the aggregated and mutated peptide, with the highest dysfunction for the mutant aggregated α-synuclein treatment.

Medical cannabis or cannabinoids for chronic non-cancer and cancer related pain: a systematic review and meta-analysis of randomised clinical trials.

OBJECTIVE: To determine the benefits and harms of medical cannabis and cannabinoids for chronic pain. DESIGN: Systematic review and meta-analysis. DATA SOURCES: MEDLINE, EMBASE, AMED, PsycInfo, CENTRAL, CINAHL, PubMed, Web of Science, Cannabis-Med, Epistemonikos, and trial registries up to January 2021. STUDY SELECTION: Randomised clinical trials of medical cannabis or cannabinoids versus any non-cannabis control for chronic pain at ≥1 month follow-up. DATA EXTRACTION AND SYNTHESIS: Paired reviewers independently assessed risk of bias and extracted data. We performed random-effects models meta-analyses and used GRADE to assess the certainty of evidence. RESULTS: A total of 32 trials with 5174 adult patients were included, 29 of which compared medical cannabis or cannabinoids with placebo. Medical cannabis was administered orally (n=30) or topically (n=2). Clinical populations included chronic non-cancer pain (n=28) and cancer related pain (n=4). Length of follow-up ranged from 1 to 5.5 months. Compared with placebo, non-inhaled medical cannabis probably results in a small increase in the proportion of patients experiencing at least the minimally important difference (MID) of 1 cm (on a 10 cm visual analogue scale (VAS)) in pain relief (modelled risk difference (RD) of 10% (95% confidence interval 5% to 15%), based on a weighted mean difference (WMD) of -0.50 cm (95% CI -0.75 to -0.25 cm, moderate certainty)). Medical cannabis taken orally results in a very small improvement in physical functioning (4% modelled RD (0.1% to 8%) for achieving at least the MID of 10 points on the 100-point SF-36 physical functioning scale, WMD of 1.67 points (0.03 to 3.31, high certainty)), and a small improvement in sleep quality (6% modelled RD (2% to 9%) for achieving at least the MID of 1 cm on a 10 cm VAS, WMD of -0.35 cm (-0.55 to -0.14 cm, high certainty)). Medical cannabis taken orally does not improve emotional, role, or social functioning (high certainty). Moderate certainty evidence shows that medical cannabis taken orally probably results in a small increased risk of transient cognitive impairment (RD 2% (0.1% to 6%)), vomiting (RD 3% (0.4% to 6%)), drowsiness (RD 5% (2% to 8%)), impaired attention (RD 3% (1% to 8%)), and nausea (RD 5% (2% to 8%)), but not diarrhoea; while high certainty evidence shows greater increased risk of dizziness (RD 9% (5% to 14%)) for trials with <3 months follow-up versus RD 28% (18% to 43%) for trials with ≥3 months follow-up; interaction test P=0.003; moderate credibility of subgroup effect). CONCLUSIONS: Moderate to high certainty evidence shows that non-inhaled medical cannabis or cannabinoids results in a small to very small improvement in pain relief, physical functioning, and sleep quality among patients with chronic pain, along with several transient adverse side effects, compared with placebo. The accompanying BMJ Rapid Recommendation provides contextualised guidance based on this body of evidence. SYSTEMATIC REVIEW REGISTRATION: https://osf.io/3pwn2.

Biodistribution of Intramuscularly-Transplanted Human Dental Pulp Stem Cells in Immunocompetent Healthy Rats through NIR Imaging.

Owing to their neural crest origin, dental pulp stem cells (DPSCs) are increasingly gaining prominence in treating nervous system disease conditions. However, as per the regulatory bodies [European-Medicines Agency (EMA), Indian-Council of Medical-Research (ICMR)], their biodistribution after transplantation needs to be evaluated for them to be considered for cell-based therapy for clinical trials. There are yet no studies describing the dynamic distribution of human origin DPSCs (hDPSCs) after transplantation in an immunocompetent, physiologically healthy animal model. Here, using near-infrared (NIR)-based whole animal and ex vivo tissue imaging, we assessed the biodistribution of intramuscularly transplanted hDPSCs in immunocompetent healthy Wistar rats. Further validation was done by quantifying gene expression of the human Alu gene in rat tissues. After 24 h of transplantation, an increase in signal intensity and area of signal was observed in the muscle of administration compared to 30 min and 6 h. At hour 24, neither increase in human Alu nor human Ki67 gene expression was seen in the rat muscle, thus confirming that the increase in signal area and intensity at hour 24 was not due to proliferation of the transplanted cells. Rather at hour 24, the NIR-signal intensity in bone marrow increased, suggesting that the NIR-tagged DPSCs have started entering into the blood vessels adjacent to the muscle, and the blood vessels being placed just beneath the subcutaneous layer might be responsible for an increase in signal intensity. Signal intensity increased distinctly in all organs at this timepoint, confirming that the cells entered the bloodstream by hour 24. Lung entrapment of DPSCs was not observed, since signal intensity was least in lungs as compared to the site of injection. Cells were retained for up to 28 days at the site of injection. These findings lay the basis to design the dosage for intramuscular delivery of hDPSCs for degenerative disease models and for future clinical trials.

Addition of corticosteroids to local anaesthetics for chronic non-cancer pain injections: a systematic review and meta-analysis of randomised controlled trials.

BACKGROUND: Despite common use, the benefit of adding steroids to local anaesthetics (SLA) for chronic non-cancer pain (CNCP) injections is uncertain. We performed a systematic review and meta-analysis of English-language RCTs to assess the benefit and safety of adding steroids to local anaesthetics (LA) for CNCP. METHODS: We searched MEDLINE, EMBASE, and CENTRAL databases from inception to May 2019. Trial selection and data extraction were performed in duplicate. Outcomes were guided by the Initiative in Methods, Measurements, and Pain Assessment in Clinical Trials (IMMPACT) statement with pain improvement as the primary outcome and pooled using random effects model and reported as relative risks (RR) or mean differences (MD) with 95% confidence intervals (CIs). RESULTS: Among 5097 abstracts, 73 trials were eligible. Although SLA increased the rate of success (42 trials, 3592 patients; RR=1.14; 95% CI, 1.03-1.25; number needed to treat [NNT], 13), the effect size decreased by nearly 50% (NNT, 22) with the removal of two intrathecal injection studies. The differences in pain scores with SLA were not clinically meaningful (54 trials, 4416 patients, MD=0.44 units; 95% CI, 0.24-0.65). No differences were observed in other outcomes or adverse events. No subgroup effects were detected based on clinical categories. Meta-regression showed no significant association with steroid dose or length of follow-up and pain relief. CONCLUSIONS: Addition of cortico steroids to local anaesthetic has only small benefits and a potential for harm. Injection of local anaesthetic alone could be therapeutic, beyond being diagnostic. A shared decision based on patient preferences should be considered. If used, one must avoid high doses and series of steroid injections. CLINICAL TRIAL REGISTRATION: PROSPERO #: CRD42015020614.

Influence of intranasal exposure of MPTP in multiple doses on liver functions and transition from non-motor to motor symptoms in a rat PD model.

Besides the effects on the striatum, the impairment of visceral organs including liver functions has been reported in Parkinson's disease (PD) patients. However, it is yet unclear if liver functions are affected in the early stage of the disease before the motor phase has appeared. The aim of our present study was thus to assess the effect of intranasal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in different doses on striatum and liver functions. Deterioration of non-motor activities appeared on single exposure to MPTP along with rise in striatum oxidative stress and decline in antioxidant levels. Decreases in dopamine, noradrenaline, and GABA and increase in serotonin were detected in striatum. Motor coordination was impaired with a single dose of MPTP, and with repeated MPTP exposure, there was further significant impairment. Locomotor activity was affected from second exposure of MPTP, and the impairment increased with third MPTP exposure. Impairment of liver function through increase in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels was observed after first MPTP insult, and it worsened with second and third administrations. First administration of MPTP triggered systemic inflammation showing significant increase in inflammatory markers in the liver. Our data shows for the first time that an intranasal route of entry of MPTP affects liver from the non-motor phase of PD itself, occurring concomitantly with the reduction of striatal dopamine. It also suggests that a single dose is not enough to bring about progression of the disease from non-motor to locomotor deficiency, and a repeated dose is needed to establish the motor severity phase in the rat intranasal MPTP model.

Okadaic Acid and Hypoxia Induced Dementia Model of Alzheimer's Type in Rats.

Alzheimer's disease (AD) is the most common cause of progressive decline of memory function in aged humans. To study about a disease mechanism and progression, animal models for the specific disease are needed. For AD, although highly valid animal models exist, none of the existing models recapitulates all aspects of human AD. The pathogenic mechanisms involved in AD are diverse and thus it is difficult to recapitulate human AD in model organisms. Intracerebroventricular (ICV) injection of okadaic acid (OKA), a protein phosphatase 2A (PP2A) inhibitor, in rats causes neurotoxicity associated with neurofibrillary degeneration. However, this model lacks amyloid pathology as observed in AD. We aimed at combining two different treatments and hence producing a better animal model of AD which may mimic most of the neuropathological, neurobehavioral, and neurochemical changes observed in AD. For this, OKA (200 ng) was microinjected bilaterally into the hippocampus of male Wistar rats followed by exposure of same rats to hypoxic conditions (10%) for 3 days. The result of which, the combination model exhibited tau hyperphosphorylation along with Aß upregulation as evident by western blotting and immunohistochemistry. The observed changes were accompanied with dysfunction of neurotransmitter system, i.e., decreased acetylcholine activity and expression. This combinatorial model also exhibited cognitive deficiency which was assessed by Morris water maze and avoidance tests along with enhanced oxidative stress which is thought to be a major player in AD pathogenesis. Taken together, we established an easily reproducible and reliable rat model for sporadic dementia of Alzheimer's type in rats which allows effective testing of new therapeutic strategies.

Opioids for Chronic Noncancer Pain: A Systematic Review and Meta-analysis.

Importance: Harms and benefits of opioids for chronic noncancer pain remain unclear. Objective: To systematically review randomized clinical trials (RCTs) of opioids for chronic noncancer pain. Data Sources and Study Selection: The databases of CENTRAL, CINAHL, EMBASE, MEDLINE, AMED, and PsycINFO were searched from inception to April 2018 for RCTs of opioids for chronic noncancer pain vs any nonopioid control. Data Extraction and Synthesis: Paired reviewers independently extracted data. The analyses used random-effects models and the Grading of Recommendations Assessment, Development and Evaluation to rate the quality of the evidence. Main Outcomes and Measures: The primary outcomes were pain intensity (score range, 0-10 cm on a visual analog scale for pain; lower is better and the minimally important difference [MID] is 1 cm), physical functioning (score range, 0-100 points on the 36-item Short Form physical component score [SF-36 PCS]; higher is better and the MID is 5 points), and incidence of vomiting. Results: Ninety-six RCTs including 26 169 participants (61% female; median age, 58 years [interquartile range, 51-61 years]) were included. Of the included studies, there were 25 trials of neuropathic pain, 32 trials of nociceptive pain, 33 trials of central sensitization (pain present in the absence of tissue damage), and 6 trials of mixed types of pain. Compared with placebo, opioid use was associated with reduced pain (weighted mean difference [WMD], -0.69 cm [95% CI, -0.82 to -0.56 cm] on a 10-cm visual analog scale for pain; modeled risk difference for achieving the MID, 11.9% [95% CI, 9.7% to 14.1%]), improved physical functioning (WMD, 2.04 points [95% CI, 1.41 to 2.68 points] on the 100-point SF-36 PCS; modeled risk difference for achieving the MID, 8.5% [95% CI, 5.9% to 11.2%]), and increased vomiting (5.9% with opioids vs 2.3% with placebo for trials that excluded patients with adverse events during a run-in period). Low- to moderate-quality evidence suggested similar associations of opioids with improvements in pain and physical functioning compared with nonsteroidal anti-inflammatory drugs (pain: WMD, -0.60 cm [95% CI, -1.54 to 0.34 cm]; physical functioning: WMD, -0.90 points [95% CI, -2.69 to 0.89 points]), tricyclic antidepressants (pain: WMD, -0.13 cm [95% CI, -0.99 to 0.74 cm]; physical functioning: WMD, -5.31 points [95% CI, -13.77 to 3.14 points]), and anticonvulsants (pain: WMD, -0.90 cm [95% CI, -1.65 to -0.14 cm]; physical functioning: WMD, 0.45 points [95% CI, -5.77 to 6.66 points]). Conclusions and Relevance: In this meta-analysis of RCTs of patients with chronic noncancer pain, evidence from high-quality studies showed that opioid use was associated with statistically significant but small improvements in pain and physical functioning, and increased risk of vomiting compared with placebo. Comparisons of opioids with nonopioid alternatives suggested that the benefit for pain and functioning may be similar, although the evidence was from studies of only low to moderate quality.

A cross-sectional study of the reporting quality of pilot or feasibility trials in high-impact anesthesia journals.

PURPOSE: Pilot trials inform the design and conduct of larger scale trials. Using the Consolidated Standards of Reporting Trials (CONSORT) pilot extension guidelines, we assessed reporting quality in five high-impact anesthesia journals and explored factors associated with reporting quality. METHODS: The five highest-impact anesthesia journals were screened for randomized-controlled trials published as pilot or feasibility trials between 2006 and 2016. A pair of reviewers independently screened citations, extracted data, and assessed reporting quality using the CONSORT pilot trial extension checklists for abstracts and full texts. We reported the percentage adherence for each item, along with the median [interquartile range (IQR)] or mean (standard deviation [SD]) for all items. The factors considered to influence reporting were: 1) trial registration, 2) industry funding, 3) trial identification as a pilot or feasibility in the title or abstract, 4) primary objective as "feasibility", and 5) the specific journal. The association was estimated using generalized estimating equations and reported as incidence rate ratios with 99% confidence intervals. RESULTS: Of 364 citations, 58 articles were eligible. The median [IQR] number of CONSORT abstract items reported was 5 [4-7], and the mean (SD) number of full text items reported was 13 (5). Significantly poor reporting was associated with "not registering the trial" (both abstracts and full texts), "trial not identified as a pilot" (abstracts), and "using clinical hypothesis as the primary objective" (full texts). CONCLUSION: The reporting quality of pilot trials published in leading anesthesia journals is poor. Journal editorial boards can encourage improved reporting by supporting adherence to the CONSORT extension for pilot trials.

Dexamethasone as an adjuvant to peripheral nerve block.

BACKGROUND: Peripheral nerve block (infiltration of local anaesthetic around a nerve) is used for anaesthesia or analgesia. A limitation to its use for postoperative analgesia is that the analgesic effect lasts only a few hours, after which moderate to severe pain at the surgical site may result in the need for alternative analgesic therapy. Several adjuvants have been used to prolong the analgesic duration of peripheral nerve block, including perineural or intravenous dexamethasone. OBJECTIVES: To evaluate the comparative efficacy and safety of perineural dexamethasone versus placebo, intravenous dexamethasone versus placebo, and perineural dexamethasone versus intravenous dexamethasone when added to peripheral nerve block for postoperative pain control in people undergoing surgery. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials, MEDLINE, Embase, DARE, Web of Science and Scopus from inception to 25 April 2017. We also searched trial registry databases, Google Scholar and meeting abstracts from the American Society of Anesthesiologists, the Canadian Anesthesiologists' Society, the American Society of Regional Anesthesia, and the European Society of Regional Anaesthesia. SELECTION CRITERIA: We included all randomized controlled trials (RCTs) comparing perineural dexamethasone with placebo, intravenous dexamethasone with placebo, or perineural dexamethasone with intravenous dexamethasone in participants receiving peripheral nerve block for upper or lower limb surgery. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane. MAIN RESULTS: We included 35 trials of 2702 participants aged 15 to 78 years; 33 studies enrolled participants undergoing upper limb surgery and two undergoing lower limb surgery. Risk of bias was low in 13 studies and high/unclear in 22. Perineural dexamethasone versus placeboDuration of sensory block was significantly longer in the perineural dexamethasone group compared with placebo (mean difference (MD) 6.70 hours, 95% confidence interval (CI) 5.54 to 7.85; participants1625; studies 27). Postoperative pain intensity at 12 and 24 hours was significantly lower in the perineural dexamethasone group compared with control (MD -2.08, 95% CI -2.63 to -1.53; participants 257; studies 5) and (MD -1.63, 95% CI -2.34 to -0.93; participants 469; studies 9), respectively. There was no significant difference at 48 hours (MD -0.61, 95% CI -1.24 to 0.03; participants 296; studies 4). The quality of evidence is very low for postoperative pain intensity at 12 hours and low for the remaining outcomes. Cumulative 24-hour postoperative opioid consumption was significantly lower in the perineural dexamethasone group compared with placebo (MD 19.25 mg, 95% CI 5.99 to 32.51; participants 380; studies 6). Intravenous dexamethasone versus placeboDuration of sensory block was significantly longer in the intravenous dexamethasone group compared with placebo (MD 6.21, 95% CI 3.53 to 8.88; participants 499; studies 8). Postoperative pain intensity at 12 and 24 hours was significantly lower in the intravenous dexamethasone group compared with placebo (MD -1.24, 95% CI -2.44 to -0.04; participants 162; studies 3) and (MD -1.26, 95% CI -2.23 to -0.29; participants 257; studies 5), respectively. There was no significant difference at 48 hours (MD -0.21, 95% CI -0.83 to 0.41; participants 172; studies 3). The quality of evidence is moderate for duration of sensory block and postoperative pain intensity at 24 hours, and low for the remaining outcomes. Cumulative 24-hour postoperative opioid consumption was significantly lower in the intravenous dexamethasone group compared with placebo (MD -6.58 mg, 95% CI -10.56 to -2.60; participants 287; studies 5). Perinerual versus intravenous dexamethasoneDuration of sensory block was significantly longer in the perineural dexamethasone group compared with intravenous by three hours (MD 3.14 hours, 95% CI 1.68 to 4.59; participants 720; studies 9). We found that postoperative pain intensity at 12 hours and 24 hours was significantly lower in the perineural dexamethasone group compared with intravenous, however, the MD did not surpass our pre-determined minimally important difference of 1.2 on the Visual Analgue Scale/Numerical Rating Scale, therefore the results are not clinically significant (MD -1.01, 95% CI -1.51 to -0.50; participants 217; studies 3) and (MD -0.77, 95% CI -1.47 to -0.08; participants 309; studies 5), respectively. There was no significant difference in severity of postoperative pain at 48 hours (MD 0.13, 95% CI -0.35 to 0.61; participants 227; studies 3). The quality of evidence is moderate for duration of sensory block and postoperative pain intensity at 24 hours, and low for the remaining outcomes. There was no difference in cumulative postoperative 24-hour opioid consumption (MD -3.87 mg, 95% CI -9.93 to 2.19; participants 242; studies 4). Incidence of severe adverse eventsFive serious adverse events were reported. One block-related event (pneumothorax) occurred in one participant in a trial comparing perineural dexamethasone and placebo; however group allocation was not reported. Four non-block-related events occurred in two trials comparing perineural dexamethasone, intravenous dexamethasone and placebo. Two participants in the placebo group required hospitalization within one week of surgery; one for a fall and one for a bowel infection. One participant in the placebo group developed Complex Regional Pain Syndrome Type I and one in the intravenous dexamethasone group developed pneumonia. The quality of evidence is very low due to the sparse number of events. AUTHORS' CONCLUSIONS: Low- to moderate-quality evidence suggests that when used as an adjuvant to peripheral nerve block in upper limb surgery, both perineural and intravenous dexamethasone may prolong duration of sensory block and are effective in reducing postoperative pain intensity and opioid consumption. There is not enough evidence to determine the effectiveness of dexamethasone as an adjuvant to peripheral nerve block in lower limb surgeries and there is no evidence in children. The results of our review may not apply to participants at risk of dexamethasone-related adverse events for whom clinical trials would probably be unsafe.There is not enough evidence to determine the effectiveness of dexamethasone as an adjuvant to peripheral nerve block in lower limb surgeries and there is no evidence in children. The results of our review may not be apply to participants who at risk of dexamethasone-related adverse events for whom clinical trials would probably be unsafe. The nine ongoing trials registered at ClinicalTrials.gov may change the results of this review.