Efficacy of reconstituted and stored botulinum toxin type A: an electrophysiologic and visual study in the auricular muscle of the rabbit

Once botulinum toxin type A is reconstituted, the manufacturer recommends that it be used in approximately 4 hours. As a result, a significant amount of this costly drug is often discarded because it is not completely used in the recommended period. The purpose of the present study was to compare fresh versus stored reconstituted botulinum toxin type A for (1) initial potency, (2) duration of action, and (3) bacterial colonization.Using a rabbit model, 20 New Zealand White rabbits were divided into four groups (I to IV). All rabbits had an injection of 2.5 U of reconstituted botulinum toxin into the right anterior auricular muscle. The first group was injected with botulinum toxin type A that was freshly reconstituted and served as the control. The second, third, and fourth groups were injected with botulinum toxin type A that had been reconstituted and stored for 2, 6, and 12 weeks, respectively, in a conventional freezer. Each rabbit had daily visual evaluation of the ear, with the position of auricle being graded from I to III. In addition, each rabbit had a nerve conduction study performed on the right anterior auricular muscle before injection and every 2 weeks after injection. Amplitude was chosen as the principal variable in the data analysis because it is the best predictor of physiologic changes at the muscle motor unit level. The endpoint of the study was defined as the time at which the nerve conduction studies and the visual inspections returned to baseline, preinjection levels. Botulinum toxin type A was also cultured before injection into each group.Overall, the nerve conduction data revealed a trend with a faster recovery (return to baseline) with the stored botulinum toxin. Groups IV and III returned to baseline first, followed by groups II and I. However, there was no significant difference among the groups at 2 and 4 weeks after injection, indicating that initial potency was unchanged. The differences between the groups became significant (p < 0.05) at 6 weeks and onward, suggesting that the duration was affected. Group I (fresh botulinum toxin) and group II (toxin stored for 2 weeks) had comparable outcomes and were not significantly different at any time period. Under visual inspection, the mean recovery time for each group was as follows: group IV, 5.4 weeks; group III, 7.0 weeks; group II, 6.75 weeks; and group I, 7.80 weeks. The results showed significance (p < 0.05) beginning after 3 weeks among some groups. Again, there was an overall quicker trend to return to baseline with the longer storage of the botulinum toxin (groups III and IV). These results support the authors' conduction study data, which suggest that the initial potency is not affected but the duration of action is. Again, groups I and II had comparable results. Microbiology cultures showed no growth of either aerobic or anaerobic bacteria at 7 days.In conclusion, using the rabbit model, it seems that reconstituted and stored botulinum toxin type A has the same initial potency but the duration of action is affected sometime after 2 weeks of storage. No bacterial contamination was associated with storing unpreserved reconstituted botulinum toxin type A for up to 12 weeks.     

The use of botulinum toxin type A in aesthetic mandibular contouring

The lower third of Asian faces is wider than that of Caucasians and it is determined by the size and width of the mandibular bone and the thickness of muscles and subcutaneous fat tissues surrounding it. Efforts to create an aesthetically slim and smooth facial contour line in nonobese people have led the authors to focus on two approaches: surgical resection of the masseteric muscle and modeling ostectomy of the square-angled mandibular bone. Because these procedures present some problems, the authors adopted a nonsurgical concept that chemically denervates muscles and reduces the bulk of the muscle. The authors have conducted a total of 1021 clinical cases from March of 2001 through September of 2002, in which patients were treated with botulinum toxin type A (Dysport; Ipsen Ltd, Slough, United Kingdom) for remodeling the lower facial contour line; 383 of those cases were followed up for at least 3 months after the initial injection. A database was made by measuring the change in the thickness of the injected muscle with an ultrasonogram. Eleven patients underwent resection of the mandibular angle before injection. The preinjection ostectomy group was involved in the study as a result of their dissatisfaction with the surgical results; they had a rather thick masseter muscle and not a bone problem. Some had both bone problems and a thick masseter muscle. Three months after the botulinum toxin injection, the thickness of the muscle was reduced by 31 percent on average. The atrophic effect of injection was observed after 2 to 4 weeks for most patients. Seventy percent of the 383 patients tracked were greatly satisfied with the result, with another 23 percent generally satisfied. No long-term side effects were reported. Masseteric hypertrophy is frequent in Asians because of racial characteristics and dietary habits. Botulinum toxin type A has made a new epoch in facial contouring for Asians. Considering that Asians have a prominent malar and a prominent mandible angle, the reduction in the thickness of the masseter can provoke relative prominence of the malar and mandible angle. Therefore, precise indication and anatomy of the facial muscle should be thoroughly understood, which will decrease the incidence of side effects and problems. Botulinum toxin type A (Dysport) injection is simple in technique, has few side effects, and promises a rapid return to daily life. The authors conclude that the injection of botulinum toxin type A can replace surgical masseter resection.     

Temporal brow lift using botulinum toxin A

The objective of this study was to determine whether brow elevation occurs as a result of paralysis of brow depressors after botulinum toxin A injection. The study's design was a prospective case series with pretreatment and posttreatment outcome evaluation with statistical analysis at a university-based division of facial plastic surgery private clinic. Twenty-two patients of a consecutive sample desiring a cosmetic enhancement underwent injection of botulinum toxin A directed to brow depressors. Injections consisted of 7 to 10 units of botulinum toxin A (Botox, Allergan, Irvine, Calif.) into selected brow depressor muscle (lateral orbicularis oculi) bilaterally. No patients withdrew for adverse effects. All patients were evaluated 2 weeks after treatment. The outcomes were measured by change in brow elevation along vertical axis extending from both midpupil and lateral canthus to the caudal row of brow hairs with eyes at neutral gaze and the head at Frankfort plane. Preintervention and postintervention brow height was measured by the primary clinical investigator. The average brow elevation from the midpupil observed after selected injection of brow depressors with botulinum toxin A was 1.02 mm (p = 0.038). The average brow elevation from the lateral canthus observed after selected injection of brow depressors with botulinum toxin A was 4.83 mm (p<0.0001). Significant temporal brow elevation occurs as the result of paralysis of brow depressors by using botulinum toxin A injection. This procedure may be considered an alternative to surgical brow elevation.     

Contralateral injections of botulinum A toxin for the treatment of hemifacial spasm to achieve increased facial symmetry.

Six patients noted facial asymmetry after botulinum toxin injection for hemifacial spasm. Each patient was injected on the side contralateral to the spasms with 10 to 15 IU over the zygomatic major and minor muscles. Each patient noted improvement in facial symmetry in the resting position and dynamic facial movements. Five of the six patients desired this approach with subsequent injections. This injection method variation proved helpful in the managing of hemifacial weakness created by botulinum A toxin for this condition.     

Acute toxicity of aminoglycoside antibiotics as an aid in detecting botulism.

Gentamicin sulfate or neomycin sulfate injected intraperitoneally into 24- to 27-g mice at a dose of 6.2 mg per mouse elicited botulism-like responses in less than 30 min, but a dose of 3.1 mg per mouse had no observable effect. The normally nontoxic 3.1-mg aminoglycoside dose aggravated the illness induced by an earlier injection of Clostridium botulinum type A or B toxin; it was usually lethal in 2 to 20 min if the preexisting illness was moderate to severe and worsened the condition of mice for about 30 min if the preexisting botulism was mild. The aminoglycoside had no effect when given shortly after the botulinum toxin was injected intraperitoneally; the sensitized state followed a latent period. It rapidly produced botulism-like effects when given to mice which had responded to a mixture of botulinum toxin and another mouse toxic agent with an illness that did not include signs of botulism. An unexpected illness devoid of botulism-like effects was encountered during intestinal colonization of mice by C. botulinum. The appearance of botulism-like signs soon after 3.1 mg of gentamicin sulfate was injected supported other suggestions that this illness included botulism that was masked by the effects of a second cause.     

Treatment of facial wounds with botulinum toxin A improves cosmetic outcome in primates

Surgeons have constantly sought to achieve the most aesthetic scar. A major factor determining the final cosmetic appearance of a cutaneous scar is the tension acting on the wound edges during the healing phase. Since Theodor Kocher pioneered the alignment of skin incisions with Langer's lines in 1892, surgical techniques that attempt to overcome closing tension have become standard. Yet, no treatment has been available to minimize underlying muscle contractions, which are the major cause of this tension. Botulinum toxin A is a potent drug that produces temporary muscular paralysis when injected locally. It has proven to be safe and effective in the treatment of a variety of disorders, including hyperkinetic facial lines. The objective of this randomized, double-blind, placebo-controlled primate study was to investigate the efficacy of a single injection of botulinum toxin A to improve the cosmetic appearance of cutaneous scars. Symmetric pairs of standardized excisions were performed on either side of the forehead of six primates. The half foreheads were randomized to the botulinum toxin A treatment side versus the placebo injection side. A panel of three blinded facial surgeons assessed the cosmetic appearance of the mature scars 3 months postoperatively. The wounds that had been immobilized with botulinum toxin A were rated as significantly better in appearance than the control wounds (p < 0.01). Histologic examination confirmed that all scars were mature. Blinded, randomized, placebo-controlled human clinical trials are presently under way at the Mayo Clinic.     

Technical delivery of myogenic cells through an endocardial injection catheter for myocardial cell implantation

BACKGROUND: The next clinical frontier in the therapeutics of ischemic heart disease may involve the development and delivery of specific molecules and cells into the myocardium. The aim of the present study was to evaluate the efficiency and safety of the MyoStar injection catheter (Biosense-Webster Inc.) that has recently been developed to deliver molecules and cells to the myocardium. The 8 Fr (110 cm length) catheter comprises a navigation sensor with a 27 gauge needle at the distal tip. METHODS: Mouse myogenic cells (C2) were delivered to a tissue culture dish through different modalities: a standard laboratory pipette, a syringe needle (27 gauge) and the injection catheter. The cells were counted and monitored for growth and differentiation in the tissue culture immediately after delivery and two, three and six days later. Cells that were injected through a regular syringe needle or through the injection catheter demonstrated the same capacity to proliferate in tissue culture up to six days. RESULTS: The behavior of the cells in culture (fusion) was identical for the cells delivered to the tissue culture by a pipette or by the injection catheter. CONCLUSION: The results of the present study indicate that delivery of cells through the MyoStar injection catheter is a method with no significant loss or adverse effects to the cells along the path of the catheter. The catheter, which possesses both injection and navigation capabilities, can be used to deliver cell therapy to patients with ischemic heart disease.     

New indications for botulinum toxin type a in cosmetics: mouth and neck

Botulinum toxin type A is frequently used to smooth hyperkinetic lines in the periocular and forehead areas of the upper face, but it has been used less frequently for indications in the lower face and neck. This study was designed to determine whether botulinum toxin treatment of the mouth and neck areas is as clinically successful as the treatment of the upper face. This was a retrospective study of patients who were treated with botulinum toxin type A (Botox) to soften hyperkinetic facial wrinkles. Of 100 patients randomly selected from a single clinical practice, 91 met the inclusion criteria and were divided into two groups for analysis. The 56 patients in group 1 did not receive treatment in the mouth and neck areas, whereas the 35 patients in group 2 were treated at least once in the mouth and neck areas. Patients were surveyed for periods ranging from 7 to 49 months. Most patients in each group had a single botulinum procedure during this period. Both groups of patients had comparable improvement of wrinkles both at the evaluation immediately after the neuromuscular blockade and during follow-up. In comparison with patients whose treatment was confined to the upper face, patients who received global treatment with botulinum toxin type A, including injections in the mouth and neck areas, were injected in more sites per procedure and had more procedures in combination with other therapies. Patient satisfaction with botulinum toxin treatment and outcomes was high in both groups. Botulinum toxin type A is an important tool within the therapeutic spectrum for the treatment of hyperkinetic facial wrinkles, including those in the areas of the mouth and neck.     

Botulinum toxin injections as a method for chemically denervating skeletal muscle to test functional hypotheses: a pilot study in Lepomis cyanellus

In this study, we demonstrate that botulinum toxin can be used to chemically denervate muscles to test functional hypotheses. We injected research-grade type A botulinum toxin complex into pectoral fin abductors (abductor superficialis) of green sunfish (Lepomis cyanellus) to determine whether chemical denervation would eliminate the ability of a particular muscle to contribute to overall pectoral fin movements. Reduction of target muscle activity occurred within 8 d of the injection, and paralysis was confirmed using electromyography. No paralysis was seen in the adjacent muscles (abductor profundus) or in positive controls (saline injections). Paralysis occurred more slowly and at lower doses than previously documented for mammals. However, botulinum toxin complex (500 kDa) was used here, whereas previous studies have used purified toxin (150 kDa). Therefore, differences in physiological responses between fish and mammals cannot yet be distinguished from differences caused by the toxin type. However, we note that the toxin complex is less likely to diffuse across muscle fascia (because it is large), which should minimize paralytic effects on adjacent muscles. We suggest that botulinum toxin holds great promise as a chemical denervation agent in functional studies of animal locomotion and feeding behaviors.     

A型肉毒素治疗带状疱疹后遗神经痛的临床观察

目的:观察A型肉毒素皮内注射治疗带状疱疹后遗神经痛(Postherpetic Neuralgia,PHN)的临床效果。方法:选择76例胸背部带状疱疹后神经痛患者,采用乒乓球抽签法随机分为观察组与对照组,每组38例患者。在口服药物治疗的基础上,观察组与对照组的局部疼痛敏感点分别皮内注射A型肉毒素与生理盐水。比较两组治疗前与治疗后第1、7、30、60天的视觉模拟评分量表(Visual Analogue Scale,VAS)及睡眠质量评分量表(Quality of Sleep,QS)评分的变化。结果:两组患者治疗后第1、7、30、60天的VAS及QS评分均明显低于治疗前(P0.05),观察组患者治疗后第1、7、30、60天的VAS及QS评分结果明显低于对照组(P0.05)。两组均未见明显的治疗相关并发症。结论:A型肉毒素皮内注射疗法可以显著减轻PHN患者的疼痛及改善睡眠质量。     

A simplified method for smile enhancement: botulinum toxin injection for gummy smile

The authors studied and provided treatment to patients with the complaint of gummy smile. Between October of 2009 and January of 2011, 52 unaesthetic smiles were evaluated and treated with onabotulinum toxin A. Botulinum toxin injection was an effective treatment, with an average satisfaction level of 9.75 on a 10-point scale. The levator labii superioris alaeque nasi is the ideal muscle for injection, and lopsided smiles could be resolved also with onabotulinum toxin A asymmetric injection. Gummy smile treatment with onabotulinum toxin A into the levator labii superioris alaeque nasi muscle is an effective method, with minimum risk of complications and very high patient satisfaction.     

A型肉毒毒素联合CO2点阵激光治疗眼周皱纹的近期疗效评估

目的:探讨A型肉毒毒素联合CO_2点阵激光治疗眼周皱纹的近期临床疗效。方法:选择2013年6月到2014年6月我院接收眼部皱纹改善手术的患者90例,随机分为激光组、肉毒毒素组及联合治疗组,各30例,分别给予CO_2点阵激光治疗、A型肉毒毒素注射和A型肉毒毒素注射联合CO_2点阵激光治疗。所有患者在治疗后7 d、1月及3月进行疗效随访评价,比较三组不良反应情况。结果:三组患者眼周皱纹均有所改善,治疗1个月时效果最为明显。激光组的静态皱纹治疗效果明显,对动态皱纹治疗效果不明显;肉毒毒素组对动态皱纹治疗效果明显,对静态皱纹治疗效果不明显;联合治疗组对静态皱纹和动态皱纹均有明显的改善,其满意度评价总分数明显高于其他两组。三组患者对CO_2点阵激光和注射A型肉毒毒素所引起疼痛均能耐受,安全性好。结论:A型肉毒毒素联合CO_2点阵激光对静态皱纹和动态皱纹均有明显的改善作用,且不良反应轻,安全性好。     

Numerical simulation of iontophoresis in the drug delivery system

The architecture and composition of stratum corneum act as barriers and limit the diffusion of most drug molecules and ions. Much effort has been made to overcome this barrier and it can be seen that iontophoresis has shown a good effect. Iontophoresis represents the application of low electrical potential to increase the transport of drugs into and across the skin or tissue. Iontophoresis is a noninvasive drug delivery system, and therefore, it is a useful alternative to drug transportation by injection. In this study, we present a numerical model and effects of electrical potential on the drug diffusion in the buccal tissue and the stratum corneum. The initial numerical results are in good comparison with experimental observation. We demonstrate that the application of an applied voltage can greatly improve the efficacy of localized drug delivery as compared to diffusion alone.     

Injection of the Arterial System of the Mouse

Cannulation of the aorta of the mouse through an incision in the left ventricle has permitted the introduction of various injection media into the arterial tree. Heparinized mice, sacrificed by ether, were exsanguinated and injected with latex, latex-thorotrast or vinyl acetate. The apparatus consists of a special cannula made by combining a 1 inch segment of a 23 gauge hypodermic needle and fine polyethylene tubing (PE 50, intramedic). Nearly all of the vascular tree was injected with this technic. Latex-thorotrast injections have the decided advantage of permitting combined X-ray and histologic study of the material. The procedure was devised in the course of an attempt to evaluate the relationship between circulatory changes and experimentally produced bone absorption and new bone formation in mice.     

Type A botulinum toxin for the treatment of hypertrophy of the masseter and temporal muscles: an alternative treatment

The treatment of hypertrophy of the masseter and temporal muscles has to date been dominated by conservative and surgical measures. Local therapy with type A botulinum toxin permits an alternative method of treatment. After targeted, sometimes electromyographically controlled, intramuscular injection of the affected muscles, marked inactivity atrophy occurred in the muscles of seven patients over the course of 3 to 8 weeks. This atrophy remained constant over a follow-up period of up to 25 months, and no side effects were observed. Because of its minimal invasiveness, this technique seems to have an advantage over conventional surgical therapy. Consequently, treatment with type A botulinum toxin can be regarded as a sensible alternative to surgery in cases of hypertrophy of the masseter and/or temporal muscles.     

Diffusion of [3H]dexamethasone in rat subcutaneous slices after injection measured by digital autoradiography

A relatively simple method for the determination of the diffusion coefficient of a substance that has been injected into tissue is described. We illustrate this method using [3]dexamethasone injected into the subcutaneous tissue of rats. Digital autoradiography was used to measure the distribution of the [3H] dexamethasone within the subcutaneous tissue at 2.5 and 20 min after injection. Measured concentration profiles of the injection were compared to a mathematical model of drug diffusion from an injection. There was good agreement between the experimental data and the mathematical model. The diffusion coefficient found using this simple injection method was (4.01 +/- 2.01) x 10(-10) m2/s. This D value was very close to the value of D = (4.11 +/- 1.77) x 10(-10) m2/s found previously using different mathematical and experimental techniques with osmotic pumps implanted for 6, 24, and 60 h in rats (1). The simple method given here for the determination of the diffusion coefficient is general enough to be applied to other substances and tissues as well.     

Activity of erythrocyte membrane Na,K-ATPase in rats with experimental botulism

The effect of type C botulinum toxin on Na, K, Mg-ATPase activities of erythrocyte membranes of white rats was studied in experiments in vivo and in vitro. The activity of Na, K, Mg-ATPase was found to be markedly inhibited in the preclinical period of poisoning, 2 hours after intraperitoneal injection of the toxin. In this case Mg-ATPase activity noticeably increased. In the presence of the development of a grave paralytic syndrome one day after intraperitoneal injection of the toxin, the activity of Na, K-ATPase of the erythrocyte membrane remained decreased as was the case in the preclinical period of poisoning, whereas the activity of Mg-ATPase returned to normal. The experiments in vitro with preincubation of erythrocyte membranes with botulinum toxin in the concentrations corresponding to the mean calculated ones in the experiments in vivo demonstrated inhibition of Na, K-ATPase. The magnitude of Mg-ATPase activity remained virtually unchanged in all the modifications of the experiments with boiled and native botulinum toxin. The in-vivo experiments with intraperitoneal injection of glutathione and unithiol to the pretreated animals attested to normalization of Na, K-ATPase in the preclinical period of poisoning, with this normalization being brought about by unithiol. In the in-vitro experiments with addition of unithiol or glutathione into the incubation medium, each of the donators of sulphhydryl groups prevented Na, K-ATPase inhibition with botulinum toxin.     

Inhibitory effects of botulinum toxin on pyloric and antral smooth muscle

Botulinum toxin injection into the pylorus is reported to improve gastric emptying in gastroparesis. Classically, botulinum toxin inhibits ACh release from cholinergic nerves in skeletal muscle. The aim of this study was to determine the effects of botulinum toxin on pyloric smooth muscle. Guinea pig pyloric muscle strips were studied in vitro. Botulinum toxin type A was added; electric field stimulation (EFS) was performed every 30 min for 6 h. ACh (100 microM)-induced contractile responses were determined before and after 6 h. Botulinum toxin caused a concentration-dependent decrease of pyloric contractions to EFS. At a low concentration (2 U/ml), botulinum toxin decreased pyloric contractions to EFS by 43 +/- 9% without affecting ACh-induced contractions. At higher concentrations (10 U/ml), botulinum toxin decreased pyloric contraction to EFS by 75 +/- 7% and decreased ACh-induced contraction by 79 +/- 9%. In conclusion, botulinum toxin inhibits pyloric smooth muscle contractility. At a low concentration, botulinum toxin decreases EFS-induced contractile responses without affecting ACh-induced contractions suggesting inhibition of ACh release from cholinergic nerves. At higher concentrations, botulinum toxin directly inhibits smooth muscle contractility as evidenced by the decreased contractile response to ACh.     

A型肉毒毒素轻链胞内持留模型的建立

[目的] 建立A型肉毒毒素轻链胞内持留模型来模拟A型肉毒毒素引起的长期中毒。[方法] 设计构建pcDNA3.1-ALC-GFP重组质粒,提取质粒进行PCR验证,并将其转染进Nureo-2a细胞中表达,利用Western Blot与细胞免疫荧光分析验证ALC-GFP的表达情况及其在细胞内的长时间持留,建立A型肉毒毒素轻链的胞内持留模型,并将该模型用于抗肉毒药物的筛选。[结果] 成功构建pcDNA3.1-ALC-GFP重组质粒并将其转染进Nureo-2a细胞中,验证了ALC-GFP在细胞内具有长时间持留性,成功建立了A型肉毒毒素轻链胞内持留模型,并利用该模型筛选出潜在抗肉毒药物CB3。[结论] 成功建立了A型肉毒毒素轻链胞内持留模型,并初步应用于CS1,CE2和CB3药物筛选,为A型肉毒毒素长期中毒的解毒研究奠定了基础。     

Intraoperative injection of botulinum toxin A into orbicularis oculi muscle for the treatment of crow's feet

The purpose of this investigation was to evaluate the degree of efficacy of eliminating crow's feet by means of direct injection of botulinum toxin A into orbicularis oculi muscles under direct surgical vision during either blepharoplasty or face lift operations. Eighteen patients were injected with Botox A-14 in each orbicularis oculi muscle. Dilution was obtained by adding 4 ml of preservative-free saline to 100 IU of Botox A. Doses ranged from 15 to 50 IU in each muscle, varying according to the severity of wrinkles and intensity of muscle contraction. In 10 patients (56 percent), the Botox was injected throughout the outer surface of both orbicularis oculi dissected during a face-lift operation. In eight other patients (44 percent), the toxin was injected into the inner surface of both orbicularis oculi exposed during classic blepharoplasty procedures. Most authors have demonstrated that the effect produced by transcutaneous Botox lasts between 4 and 6 months; the paralysis obtained by direct muscular injection was effective for 9 months in 14 patients (78 percent) and 10 months in the other 4 patients (22 percent). Results were documented by means of preinjection and postinjection photographs, videotapes, and electromyographs. Neither local nor general adverse effects were noted. The improvement obtained in crow's feet was satisfactory to the patient and to us. The use of Botox intraoperatively permitted at the same time not only the treatment of crow's feet by paralysis of orbicularis oculi muscles but also the correction of senile changes in the lids and face by means of either blepharoplasty or face-lift operations.