Connexin Hemichannels: Methods for Dye Uptake and Leakage.

It is now clear that connexin-based, gap junction "hemichannels" in an undocked state are capable of opening and connecting cytoplasm to the extracellular milieu. Varied studies also suggest that such channel activity plays a vital role in diverse cell processes and abnormal hemichannel activity contributes to pathogenesis. To pursue fundamental questions in this area, investigators require methods for studying hemichannel permeability and dynamics that are quantitative, sensitive, versatile, and available to most cellular and molecular laboratories. Here we first provide a theoretical background for this work, including the role of cellular membrane potentials. We then describe in detail our computer-assisted methods for both dye uptake and leakage along with illustrative results from different cell systems. A key feature of our protocol is the inclusion of a mechanical stimulation step. We describe dye uptake, interpreted as connexin dependent, that is shown to be enhanced with reduced extracellular Ca2+, mechanically responsive, inhibited by TPA, inhibited by EL186 antibodies for Cx43 and sustained for more than 15 min following mechanical stimulation. We describe dye leakage that displays these same properties, with estimates of hemichannel numbers per cell being derived from leakage rates. We also describe dye uptake that is shown to be unaffected by a reduction in external Ca2+, insensitive to EL186 antibodies and relatively short-lived following mechanical stimulation; this uptake may occur via pannexin 1 channels expressed in the cells studied here. It is unlikely that cell damage plays a significant role in dye uptake following mechanical stimulation, given compelling results from various control experiments.

Botulinum toxin for shoulder pain.

BACKGROUND: Recent evidence suggests an anti-nociceptive effect of botulinum toxin. OBJECTIVES: To compare the efficacy and safety of botulinum toxin in comparison to placebo or other treatment options for shoulder pain. SEARCH STRATEGY: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library), Ovid MEDLINE, CINAHL (via EBSCOhost), Ovid SPORTDiscus, EMBASE and Science Citation Index. SELECTION CRITERIA: Randomized controlled trials (RCTs) comparing botulinum toxin with placebo or active treatment in people with shoulder pain were included. DATA COLLECTION AND ANALYSIS: For continuous measures we calculated mean difference (MD), and for categorical measures risk ratio (RR) (with 95% confidence interval (CI)). MAIN RESULTS: Six RCTs with 164 patients were included. Five RCTs in participants with post-stroke shoulder pain indicated that compared with placebo, a single intramuscular injection of botulinum toxin A significantly reduced pain at three to six months post-injection (MD -1.2 points, 95% CI -2.4 to -0.07; 0 to 10 point scale) but not at one month (MD -1.1 points, 95% CI -2.9 to 0.7). Shoulder external rotation was increased at one month (MD 9.8 degrees , 95% CI 0.2 to 19.4) but not at three to six months. Shoulder abduction, external rotation or spasticity did not differ between groups, nor did the number of adverse events (RR 1.46, 95% CI 0.6 to 24.3).One RCT in arthritis-related shoulder pain indicated that botulinum toxin reduced pain severity (MD -2.0, 95% CI -3.7 to -0.3; 10 point scale) and shoulder disability with a reduction in Shoulder Pain and Disability Index score (MD -13.4, 95% CI -24.9 to -1.9; 100 point scale) when compared with placebo. Shoulder abduction was improved (MD 13.8 degrees, 95% CI 3.2 to 44.0). Serious adverse events did not differ between groups (RR 0.35, 95% CI: 0.11, 1.12). AUTHORS' CONCLUSIONS: The results should be interpreted with caution due to few studies with small sample sizes and high risk of bias. Botulinum toxin A injections seem to reduce pain severity and improve shoulder function and range of motion when compared with placebo in patients with shoulder pain due to spastic hemiplegia or arthritis. It is unclear if the benefit of pain relief in post-stroke shoulder pain at three to six months but not at one month is due to limitations of the evidence, which includes small sample sizes with imprecise estimates, or a delayed onset of action. More studies with safety data are needed.

Botulinum toxin for shoulder pain: a cochrane systematic review.

OBJECTIVE: To perform a Cochrane Systematic Review of benefits and harms of botulinum toxin for shoulder pain. METHODS: We included clinical trials of adults with shoulder pain (population), comparing botulinum toxin (intervention) to placebo or other therapies (comparison), and reporting benefits or harms (outcomes). We calculated relative risk (RR) for categorical outcomes and mean differences (MD) for continuous outcomes. RESULTS: Six randomized controlled trials (RCT) with 164 patients all comparing single botulinum toxin type A injections to placebo were included. Five RCT in patients with post-stroke shoulder pain found that an intramuscular injection of botulinum toxin type A significantly reduced pain at 3-6 months (MD -1.2 points on 0-10 scale, 95% CI -2.4 to -0.07) and improved shoulder external rotation at 1 month (MD 9.8°, 95% CI 0.2° to 19.4°). Number of adverse events did not differ between groups (RR 1.46, 95% CI 0.6 to 24.3). One RCT in arthritis-related shoulder pain showed that single intraarticular botulinum toxin type A injection reduced pain (MD -2.0 on 0-10 scale, 95% CI -3.7 to -0.3) and shoulder disability (MD -13.4 on 0-100 scale, 95% CI -24.9 to -1.9) and improved shoulder abduction (MD 13.8°, 95% CI 3.2° to 44.0°) at 1 month, compared with placebo. Serious adverse events did not differ between groups (RR 0.35, 95% CI 0.11, 1.12). CONCLUSION: With evidence from few studies with small sample sizes and medium to high risk of bias, botulinum toxin type A injections decreased pain and improved shoulder function in patients with chronic shoulder pain due to spastic hemiplegia or arthritis.