Neurotoxins produced by the gram-positive anaerobic bacterium Clostridium botulinum are the most potent toxins known to mankind and are the causative agents of botulism. Seven distinct botulinum toxins (botulinum toxin types A, B, C, D, E, F, and G) produced by different strains of C. botulinum have been described (1). The human nervous system is susceptible to five toxin serotypes (botulinum toxin types A, B, E, F, G) and unaffected by two (botulinum toxin types C, D) (2). All serotypes act preferentially on the peripheral nervous system where they inhibit release of acetylcholine from the presynaptic terminal of the neuromuscular junction (3). At higher doses, toxins may bind to nerve terminals at autonomic cholinergic ganglia with autonomic effects (4). Botulinum toxin also may have inhibitory effects on the release of other neurotransmitters such as substance P (5).

Botulinum toxin’s mechanism of action includes a three-step process: binding, internalization, and neuromuscular blockade. With botulinum toxin type A, clinical expression of the effects typically takes 24 to 48 hours, and maximal muscle weakening is not seen for 1 week. The first step is the irreversible binding of botulinum toxin type A to presynaptic cholinergic receptors via the H chain’s 50-kDa carboxy-terminal (6, 7 and 8). The second step involves internalization of the neurotoxin through a receptor-mediated endocytosis (9). The third step is neuromuscular blockade. Within the synapse, isoforms of proteins form a complex platform for docking and fusion of acetylcholine vesicles to the cell membrane that is necessary prior to release (10,11). These protein isoforms are vesicle-associated membrane protein (VAMP, also known as synaptobrevin), synaptosomal associated protein (SNAP-25), and syntaxin. Alteration of these membrane proteins prevents the binding of the vesicles that release the acetylcholine. The botulinum toxin serotypes act in different locations on these docking proteins. The more profoundly the membrane proteins are structurally altered, the sooner they are replaced and the faster the effects of the toxin abate. This may explain some of the difference in duration of effects of the different serotypes. While still inactivating the docking process, the longer-acting serotypes may not alter the molecule significantly enough that the cell immediately recognizes that it must be replaced.

In the United States, there are two commercially available botulinum toxin preparations: (i) botulinum toxin type A (Botox, Allergan, Irvine, CA) and (ii) botulinum toxin type B, Myobloc (Myobloc, Elan Pharmaceuticals, San Diego, CA). Dysport (Ipsen Ltd., Berkshire, United Kingdom), a second preparation of botulinum toxin type A, currently is not available in the United States. The two FDA-approved formulations of botulinum toxin type A are different and not equivalent in dosing. The Botox unit is three to five times as potent as the Dysport unit (12), but this conversion ratio does not take into consideration safety or antigenic potential (13). Comparable dosing for Myobloc and Botox is still under investigation but may be in the range from 50 to 150 U of Myobloc to 1U of Botox.

Botulinum toxins are measured in mouse units (MU) or (U). The mouse biologic assay is currently the only accepted quantitative method for the detection of clostridium toxins in culture, serum, and food samples (14), and for antitoxin standardization (15). It is the most sensitive and specific measurement of botulinum toxin activity. One mouse unit is defined as the median intraperitoneal dose
required to kill 50% of a batch of 18- to 20-g female Swiss-Webster mice (LD₅₀) over 3 to 4 days (16, 17 and 18).

The currently available formulation of Botox, derived from toxins prepared by Allergan, Inc. in 1997, has replaced the old batch originally prepared by Schantz in 1979 (19). In the Schantz preparation, 1 MU of the crystalline protein complex weighed about 0.043 ng (17). The amount of chromatographically purified botulinum A toxin was approximately 0.006 ng (20). The new batch has only 20% of the protein content of the materials sold prior to 1997, but an equivalent 100 U dose of toxin. No significant alteration in dosing was recommended with the new preparation, but the lower protein load may decrease the immunogenicity of the product. Botox is a sterile, lyophilized (vacuum-dried) form of purified botulinum toxin type A, produced from a culture of the Hall strain of C. botulinum. It is isolated from the culture solution by a series of acid precipitations to a crystalline complex consisting of the active high-molecular-weight toxin protein and an associated hemagglutinin protein. The crystalline complex is redissolved in a solution containing saline and albumin for stability, and sterile filtered prior to vacuum drying.

Each vial of Botox contains 100 MU of C. botulinum toxin type A (with 10% variability), 0.5 mg of human albumin, and 0.9 mg of sodium chloride in a sterile, vacuum-dried form without a preservative. The vials are stored in the freezer before reconstitution for clinical use. The recommended dilutant is nonpreserved normal saline. Whether use of preserved saline during reconstitution alters the dose, response, discomfort at the time of injection, or duration of response (21) is under investigation. After reconstitution, the product should be stored in the refrigerator at 2 to 8°C (22). Some reports suggest that the material will retain potency beyond the manufacturer recommended 4 hours, and that this effect may be prolonged by refrigeration of the solution (23). Other studies show a loss of function with refrigeration (24). The concentration of Botox solution is dependent on the volume of saline added and typically is described by how many units are present in 0.1 mL. For cosmetic uses, the reported concentrations of the solution vary from 1.0 U per 0.1 mL to 10 U per 0.1 mL. The reported volumes of solutions used for cosmetic indications vary from 0.025 to 1.0 mL per site. The effect of botulinum toxin type A is dependent on the location, concentration, and volume of solution that is injected.

Dysport (Ipsen Ltd., United Kingdom) (botulinum toxin type A) preparation is different in terms of mouse units, chemical properties, biologic activities, and weight (25). It is supplied in 500-MU vials, produced by column-based purification rather than by the precipitation technique used for Botox, and can be stored at room temperature. The recommended dilutant also is unpreserved normal saline (26). For equivalent effect, dose is multiplied by three to five times the units of Botox one would use. Dysport has been used successfully for blepharospasm (25), torticollis (27), hemifacial spasm (28), and hyperfunctional facial lines (26).

Myobloc, the only commercially available preparation of botulinum type B, is supplied in a sterile injectable solution in 3.5-mL glass vials. The vials are available in volumes of 2,500, 5,000, and 10,000 U. The botulinum type B is in solution with 0.05% human serum albumin, 0.01 M sodium succinate, and 0.1 M sodium chloride at pH 5.6. The bottles are all ready-to-use solutions and do not require reconstitution. Myobloc is stable for 21 months if stored in the refrigerator. If Myobloc is diluted or buffered, the product insert recommends that it be used within 4 hours. Myobloc is FDA approved for use in cervical dystonia (29).

Myobloc can be buffered with sodium bicarbonate to decrease the pain of the injections. The biologic effect of the buffering has not yet been established with respect to diffusion, efficacy, or alteration of the duration of effects. The cosmetic equivalency of botulinum types A and B are still being evaluated and at this time are anecdotal rather than scientifically established. Ratios of 25:150 U of Myobloc for every 1 U of Botox have been described (30,31). To achieve the higher doses,
larger volumes of the undiluted Myobloc solution must be injected than those typically used with Botox. This also may alter diffusion. At current pricing levels, Myobloc becomes more expensive than Botox when the ratio exceeds 50:1.

Botulinum toxin is administered by injection into the target muscle. To choose the site of injection to treat wrinkles, the patient is asked to squeeze and relax the muscles in the affected area. The surgeon identifies the location of maximal skin displacement during the contraction of the muscle. The solution is injected at that point and into the muscle rather than into the crease line. Unlike “filler” techniques, the Botox should be aimed at the muscle rather than the crease to be most effective. The injection should go into the muscle layer or into the subdermal tissue just above the muscle in the areas where the facial skin is thin. The muscles are relaxed prior to injection to decrease the pain of the injection. The patient experiences the discomfort of the needle stick, followed by a localized “stinging” as the solution is injected. There may be some pressure sensation from the volume of the fluid injected. The onset of action is variable from patient to patient and different from one injection to another in the same patient, but most will notice alteration in muscle contraction within 24 to 48 hours. In research studies, the maximal response in muscle weakness does not occur for 7 days. The effects of Botox are temporary and resolve between 2 and 11 months after the injection. In subsequent injections, injections of the same volume, concentration, and location may have a different duration of effect in the same person. Resolution of the paresis is believed to occur when the neurons develop alternative terminals for neurotransmission, and when the presynaptic neuron replaces the altered transporter proteins. The rate at which these actions occur may partially explain the variability in the duration of effects. It still is uncertain whether cosmetic patients will develop tachyphylaxis to botulinum toxin. In cases where botulinum toxin type A has been used for long-term treatment of dystonic muscle disorders, some reviews indicate a “resistance” develops, whereas other reports show no long-term decrease in efficacy (21). In contrast to this, anecdotal reports on the cosmetic use suggest that patients who have had multiple injections over a number of years may enjoy a lengthening of the duration of the effects.

When the patient begins to notice the changes from Botox injection, there are two phases to the response. The early alteration in the dynamic wrinkle lines comes from a relaxation of the resting tone to the muscle, decreased force of contraction, and perhaps shift in the tissue fluids. This occurs in the first week. The second and more chronic process is remodeling of the dermis that occurs when the mechanical pressure of the contraction is relaxed. In addition to decreasing the wrinkle lines that are present, prolonged use of botulinum toxin should prevent further deepening of the creases. This makes Botox one of the few modalities that will truly prevent visible signs of aging. The most dramatic responses to treatment are seen in patients in the age range from 30 to 50 years. In these cases, the injections may obliterate the kinetic lines. With deeper wrinkles, treatment with botulinum toxin will flatten the edges of the indentation, but additional filler techniques are usually necessary to make the area smooth. Botulinum toxin type A may be used in association with collagen, polytetrafluoroethylene, silicone, hyaluronic acid preparations, and other materials that mechanically elevate the depressed tissue of the wrinkle line.

Preinjection counseling should cover the potential side effects of the botulinum toxin type A injections, as well as highlight the beneficial aspects of treatment. The side effects may be localized effects of the injection or may occur when there is
inadvertent spread of the Botox to the surrounding facial neuromuscular junctions. Small hematomas may occur when blood vessels are inadvertently injured by the injection needle. To help prevent this, digital pressure is placed on the injection site when the needle is withdrawn. The volume of the injection and the location of the injection in the perioral region may lead to decreased strength of oral closure, lip droop, and drooling. Although many of these complications have not yet been reported in the cosmetic patients, they have been seen in patients who receive therapeutic injections. The incidence of unplanned spread of the neurotoxin in the cosmetic patients is low. Of these, postinjection ptosis has been reported most frequently. Temporary redness at the injection site is common. Flu-like syndrome and headache have been noted. The albumin in Botox is a human product, and although there has never been any evidence of contamination or disease transmission, discussion of these concerns may occur.

Botulinum toxin has been used for glabellar folds, lateral periocular rhytides, lower eyelid orbicularis ridges, brow elevation, horizontal forehead wrinkles, perioral lipstick lines, melolabial folds, and platysmal bands in the neck. It can be used as a singular therapy or in conjunction with other cosmetic surgical interventions. Botulinum toxin can be used to augment the results of laser resurfacing, chemical peels, wrinkle filler techniques, endoscopic forehead surgery, and lower eyelid blepharoplasty.

If 10 expert physician Botox injectors are asked to describe injection techniques, 10 different answers will be received. Total dose, solution concentration, location of injection, and depth of injection are just four examples of variables that can be altered. The size of the patient’s muscles, the degree of facial dynamic action, previous response to injection, and desired extent of post injection paresis are factored into an individual dosing and injection pattern. Despite all of these variabilities, certain tenants remain constant. Until the synapses are saturated, increasing the dose increases the degree of paresis. Increasing either the total dose or the volume of injection at a given location will increase diffusion. The molecule has its effect at the neuromuscular junction, so it will be more effective when injected at or near this site.

Dilution techniques and injection materials also vary. Botox is supplied in bottles of 100 U, and the amount of saline added to the bottle determines the concentration of the solution. The product insert recommends unpreserved saline for dilution, but recent studies have suggested that preserved saline may cause less discomfort with injection and that the preservative chemicals do not disrupt the action of the botulinum toxin molecule (21). Some treatment techniques use the same concentration of solution at all locations, but other techniques vary the concentration at different locations. This is accomplished by altering the dilution in the bottle or by changing the concentration in the syringe. Altering the concentration in the syringe may result in uneven dilution, but this has not been found to be clinically troublesome. Injections may be given with a 1-mL syringe with a 30-gauge needle or with a one-piece 1-mL insulin syringe. Insulin syringes have the advantage of less wasted material in the needle hub but have the disadvantage that the needle cannot be changed if it becomes dull or contaminated.

Skin preparation varies from no preparation to 45-minute pretreatment with topical anesthetic and sterile cleansing techniques. For most cosmetic patients, the discomfort of the injections does not warrant the time necessary for the topical aesthetic to work, but for some it makes the injections less unpleasant. Alcohol may denature the botulinum toxin, so for that reason, some physicians prefer to use povidone iodine (Betadine) to clean the skin. A marking pen can be used to identify
the locations of the injections. This helps the physician track the locations while injecting, assists the scribe in noting the locations in the chart, and (using a mirror) allows the repeat patient to participate in location selection based on previous response. Many cosmetic patients are discriminating observers and will appreciate the opportunity to influence the design of their treatment pattern.

Dosing and concentration information in the treatment techniques described in this chapter are based on Botox. This information can be altered to apply Dysport or Myobloc once appropriate dosing ratios have been determined.