The Value of Injection Therapy with Botulinum Toxin in Pain Treatment of Primary Chronic Anal Fissures Compared to Anal Dilation, and Local Nifedipine in Combination with Lidocaine.

Introduction: Anal fissure is a longitudinal tear of the mucosa of the anal canal extending from the outer anal orifice in the direction of the dentate line of the inner anal opening. Fissures are divided into primary and secondary, and acute or chronic. Besides minimal rectal bleeding, itching and soiling, primary chronic anal fissures (PCAF) manifest with anal pain as theirs main determinant. It is described as the most troubling symptom. Aim: To compare the effect of injection therapy with botulinum toxin A (ITBT) vs. anal dilation (AD), and local nifedipine with lidocaine (LNL) in pain treatment of PCAF. Materials and Methods: This controlled retrospective prospective longitudinal study covered 94 patients, divided in 3 groups. The first was treated with ITBT, the second with AD and third using LNL (31, 33 and 30 patients respectively). Clostridium botulinum toxin A was used, dissolved with saline to concentration of 200 U/ml. The solution was applied to both sides of PCAF at dose of 40U. Modified technique of AD was done using 3 fingers of a single hand, progressively introduced into the anal canal, followed by gradual lateral distraction during 1 min. LNL therapy was conducted using nifedipine (0.3%) with lidocaine (1.5%) ointment, applied twice daily for 3 weeks. To measure pain, a visual analog scale (VAS) was used. The follow-up period was 12 weeks with checkup at week 4. Results: The median age of participants was 46.6±13.9 years (50 males vs. 44 females). The type of therapy had a significantly different effect on pain at week 4 (p=0.0003). Severe pain was present in only 2 ITBT patients, 16 AD, and 6 LNL patients. Post hoc analyses showed different pain disappearance time by week 12 (p <0.0001). The mean time was shortest in ITBT group (6.1±1.5 weeks). Anal pain intensity significantly differed among the 3 groups (Fisher exact, p=0.002). Namely, 71% in ITBT group rated the pain as weakest (VAS score 1) compared to 18.2% in AD and 30% of patients in LNL group. The overall pain reduction significance was in favor of ITBT, due to the differences between the ITBT and AD groups (p=0.00024) and ITBT compared to LNL group (p=0.018). Conclusion: ITBT is superior to AD and LNL in reducing pain in PCAF.

Lentiviral vector-mediated expression of C3 transferase attenuates retinal ischemia and reperfusion injury in rats.

AIMS: Our previous study showed that intravitreal delivery of self-complementary AAV2 (scAAV2)-mediated exoenzyme C3 transferase (C3) can attenuate retinal ischemia/reperfusion (I/R) injury. The current study investigated the neuroprotective effects of lentivirus (LV)-mediated C3 transgene expression on rat retinal I/R injury. MAIN METHODS: The LV encoding C3 and green fluorescent protein (GFP) together (LV-C3-GFP) or GFP only (LV-GFP) was intravitreally injected to SPRAGUE-DAWLEY rats. On day 5 post-intravitreal injection, eyes were evaluated by slit-lamp examination. The GFP expression on retina was confirmed by in vivo and ex vivo assessments. RhoA GTPase expression in retina was examined by western blot. Retinal I/R injury was generated by transiently increasing intraocular pressure (110 mmHg, 90 min). Eyes were then enucleated, and retinas processed for morphological analysis and TdT-dUTP terminal nick-end labeling (TUNEL) assay. KEY FINDINGS: No obvious inflammatory reactions or surgical complications were observed after intravitreal injection of LV vectors. There was a significant decrease of total RhoA GTPase level in the retina treated with LV-C3-GFP. Compared to the blank control group, LV-C3-GFP and LV-GFP did not affect the retinal thickness, cell density in ganglion cell layer (GCL), or numbers of apoptotic cells in retinal flat-mounts. In the LV-GFP-treated retinas, I/R decreased the retinal thickness and GCL cell density and increased apoptotic retinal cell numbers. LV-C3-GFP significantly protected against all these degenerative effects of I/R. SIGNIFICANCE: This study indicated that LV-mediated C3 transgene expression exhibits neuroprotective effects on the retinal I/R injury and holds potential as a novel neuroprotective approach targeting certain retinopathies.

The Effect of Botulinum Toxin on Flap Viability of the Posterior Thigh Perforator Flap in Rats.

BACKGROUND: The use of perforator propeller flaps in lower limb reconstruction has increased recently. Many pharmacological agents are used to increase flap viability. Botulinum toxin has been used in various types of flaps in the literature. However, there is no study regarding the use of botulinum toxin in the lower limb propeller flaps. This study investigates the effect of botulinum toxin administration on flap survival for lower limb propeller flap in rats. MATERIALS AND METHODS: The study included 20 male Wistar albino rats, divided into two groups with a flap rotation of 90° in group 1 and 180° in group 2. In both groups, botulinum toxin was administered to the right thigh and a physiological saline solution was applied to the left thigh. Five days later, flaps were elevated over the posterior aspect of the right and left thighs and inset after 90° and 180° rotation was performed. Histopathological, immunohistochemical, and necrosis area analyses were performed. RESULTS: Necrosis area, edema, polymorphonuclear leukocyte infiltration, and necrosis were found to be higher on the left side of the groups, whereas epidermal thickness, collagen density, vascularization, and hair root density were found to be higher on the right side of the groups. No significant difference was found between the right posterior thighs in either group on any parameter other than vascularization. Histopathologically and immunochemically statistically significant differences were found between the two groups. CONCLUSIONS: The present study found that botulinum toxin increases flap viability in lower limb perforator-based propeller flaps.

Astragaloside IV attenuates sepsis-induced intestinal barrier dysfunction via suppressing RhoA/NLRP3 inflammasome signaling.

Intestinal barrier dysfunction is a trigger for sepsis progression. NLRP3 inflammasome and RhoA contribute to sepsis and intestinal inflammation. The current study aimed to explore the effects of Astragaloside IV (AS-IV), a bioactive compound from Astragalus membranaceus, on sepsis-caused intestinal barrier dysfunction and whether NLRP3 inflammasome and RhoA are involved. Septic mice modeled by cecal ligation and puncture (CLP) operation were administered with 3 mg/kg AS-IV intravenously. AS-IV decreased mortality, cytokines release, I-FABP secretion, intestinal histological score and barrier permeability, and increased tight junction (TJ) expression in intestine in CLP model. Also, in Caco-2 cells subjected to lipopolysaccharide (LPS), 200 µg/mL AS-IV co-incubation reduced cytokines levels and enhanced in vitro gut barrier function without cytotoxicity. Subsequently, NLRP3 inflammasome and RhoA were highly activated both in intestinal tissue in vivo and in Caco-2 cells in vitro, both of which were significantly suppressed by AS-IV treatment. In addition, the benefits of AS-IV on Caco-2 monolayer barrier were largely counteracted by RhoA agonist CN03 and NLRP3 gene overexpression, respectively. Furthermore, LPS-induced NLRP3 inflammasome activation was abrogated by RhoA inhibitor C3 exoenzyme. However, NLRP3 knockdown by siRNA hardly affected RhoA activation in Caco-2 cells. These data suggest that AS-IV protects intestinal epithelium from sepsis-induced barrier dysfunction via inhibiting RhoA/NLRP3 inflammasome signal pathway.

Surgical effect and electrophysiological study of patients with hemifacial spasm treated with botulinum toxin or acupuncture before microvascular decompression.

OBJECTIVE: We investigated patients with hemifacial spasm (HFS) who received a botulinum toxin (BT) injection or acupuncture before receiving microvascular decompression (MVD) to determine whether it affects the success rate of surgery. Abnormal Muscle Response (AMR) and Compound Motor Action Potential (CMAP) are commonly used as electrophysiological monitoring methods in surgery, and we will compare the differences between these patients in this regard. PATIENTS AND METHODS: A total of 539 patients with HFS underwent MVD treatment in our department between January 2014 and June 2017. Among them, 83 patients had received BT injection before surgery and were recorded as BT group. Eighty-three patients underwent acupuncture before surgery and were recorded as acupuncture group. Five patients received both BT injection and acupuncture before surgery and were recorded as mixed group. A total of 368 patients who had not received any treatment before surgery were recorded as simple MVD group. We calculated the immediate and long-term remission rates after surgery. AMR and CMAP monitoring were routinely performed during surgery. RESULTS: Immediate remission rate after surgery was 96.4% (80/83) in BT group, 100% (83/83) in acupuncture group, 100% (5/5) in mixed group, and 95.1% (350/368) in simple MVD group, and the immediate remission rate of BT group is significantly higher than that of simple MVD group (p = 0.04). Long-term remission rate: the remission rates of the four groups were 94.0% (78/83), 97.6% (81/83), 100.0% (5/5) and 92.7%(341/368), respectively, and there is no statistical difference between them (p > 0.05). The amplitude of one branch or several branches of CMAP on the affected side was lower than the healthy side in BT or acupuncture treatment patients. CONCLUSIONS: A preoperative BT injection or acupuncture treatment do not reduce the postoperative remission rate of HFS patients treated with MVD, and the amplitude of CMAP on the affected side was lower than the healthy side.

A SNAP-25 cleaving chimera of botulinum neurotoxin /A and /E prevents TNFα-induced elevation of the activities of native TRP channels on early postnatal rat dorsal root ganglion neurons.

Transient receptor potential (TRP) vallinoid 1 (TRPV1) and ankyrin 1 (TRPA1) are two transducing channels expressed on peripheral sensory nerves involved in pain sensation. Upregulation of their expression, stimulated by inflammatory cytokines and growth factors in animal pain models, correlate with the induction of nociceptive hyper-sensitivity. Herein, we firstly demonstrate by immuno-cytochemical labelling that TNFα augments the surface content of these channels on rat cultured dorsal root ganglion (DRG) neurons which, in turn, enhances the electrophysiological and functional responses of the latter to their specific agonists. A molecular basis underlying this TNFα-dependent enhancement was unveiled by pre-treating DRGs with a recently-published chimeric protein, consisting of the protease light chain (LC) of botulinum neurotoxin (BoNT) serotype E fused to full-length BoNT/A (LC/E-BoNT/A). This cleaves synaptosomal-associated protein of Mr 25k (SNAP-25) and reported previously to exhibit anti-nociceptive activity in a rat model of neuropathic pain. Low pM concentrations of this chimera were found to prevent the TNFα-stimulated delivery of TRPV1/A1 to the neuronal plasmalemma and, accordingly, decreased their incremental functional activities relative to those of control cells, an effect accompanied by SNAP-25 cleavage. Advantageously, LC/E-BoNT/A did not reduce the basal surface contents of the two channels or their pharmacological responses. Thus, use of multiple complementary methodologies provides evidence that LC/E-BoNT/A abolishes the TNFα-dependent augmented, but not resting, surface trafficking of TRPV1/A1. As TNFα is known to induce nociceptive hyper-sensitivity in vivo, our observed inhibition by LC/E-BoNT/A of its action in vitro could contribute to its potential alleviation of pain.

Long-Term High-Dose Resveratrol Supplementation Reduces Bone Mass and Fracture Strength in Rats.

Resveratrol (RSV) is a natural polyphenolic compound. A recent study suggests a positive effect on BMD in men; however, the underlying changes in microstructure and strength remain unknown. We aimed to investigate the effects of RSV on the skeleton in hindlimb-immobilized and non-immobilized rats. Seventy-two female Wistar rats were divided into six groups. Two baseline (BSL) groups underwent short-term diet intervention for 4 weeks before sacrifice [phytoestrogen-deficient diet (PD) (BSL + PD) or RSV diet (600 mg/kg body weight/day) (BSL + RSV)]. Four groups were injected in the right hindlimb with botulinum toxin (BTX) (immobilized) or saline (non-immobilized), and fed either PD diet or RSV diet 4 weeks pre-injection and 6 weeks post-injection before sacrifice (BTX + PD, BTX + RSV, PD, and RSV, respectively). DXA, µCT, dynamic histomorphometry, and mechanical tests were performed. Short-term RSV treatment did not affect bone parameters, whereas long-term RSV exposure had a consistent negative impact on non-immobilized rats (RSV vs. PD); whole femoral aBMD (p = 0.01) and distal femoral metaphyseal Tb.N (p = 0.01), Tb.Sp (p = 0.02), and BV/TV (p = 0.07). At the femoral mid-diaphysis, RSV increased periosteal resorption (p = 0.01) and increased endosteal formation (p = 0.02), while mineralization was unaffected. In addition, RSV reduced femoral mid-diaphyseal three-point bending strength (p = 0.03) and stiffness (p = 0.04). BTX-induced immobilization resulted in significant bone loss and reduced bone strength; however, RSV supplementation was unable to prevent this. In conclusion, long-term high-dose RSV reduced bone mass and fracture strength and did not prevent immobilization-induced bone loss in rats.

Human Induced Pluripotent Stem Cell Derived Neuronal Cells Cultured on Chemically-Defined Hydrogels for Sensitive In Vitro Detection of Botulinum Neurotoxin.

Botulinum neurotoxin (BoNT) detection provides a useful model for validating cell-based neurotoxicity screening approaches, as sensitivity is dependent on functionally competent neurons and clear quantitative endpoints are available for correlating results to approved animal testing protocols. Here, human induced pluripotent stem cell (iPSC)-derived neuronal cells were cultured on chemically-defined poly(ethylene glycol) (PEG) hydrogels formed by "thiol-ene" photopolymerization and tested as a cell-based neurotoxicity assay by determining sensitivity to active BoNT/A1. BoNT/A1 sensitivity was comparable to the approved in vivo mouse bioassay for human iPSC-derived neurons and neural stem cells (iPSC-NSCs) cultured on PEG hydrogels or treated tissue culture polystyrene (TCP) surfaces. However, maximum sensitivity for BoNT detection was achieved two weeks earlier for iPSC-NSCs that were differentiated and matured on PEG hydrogels compared to TCP. Therefore, chemically-defined synthetic hydrogels offer benefits over standard platforms when optimizing culture conditions for cell-based screening and achieve sensitivities comparable to an approved animal testing protocol.

Explanation of timing of botulinum neurotoxin effects, onset and duration, and clinical ways of influencing them.

While the steps in the action of botulinum neurotoxin (BoNT) are well known, the factors underlying the timing of these steps are not fully understood. After toxin is injected into a muscle, it resides in the extracellular space and must be taken up into the nerve terminals. More toxin will be taken up if near the endplate. Toxin is distributed mainly by convection and there is likely little diffusion. Toxin that is not taken up will go into the general circulation where it may have a slight systemic effect. The uptake is activity and temperature dependent. Encouraging the unwanted muscle contractions after injection should be helpful. Cooling will decrease the uptake. The times for washout from the extracellular space and uptake of the toxin are not well established, but are likely measured in minutes. Toxin in the general circulation has a long half time. The time from injection to weakness is determined by how long it takes to get sufficient damage of the SNARE proteins to interfere with synaptic release. Toxins are zinc dependent proteases, and supplemental zinc may produce a greater effect. There will be weakness as long as there is residual toxin in the nerve ending.

Studying Axonal Outgrowth and Regeneration of the Corticospinal Tract in Organotypic Slice Cultures.

Studies of axonal outgrowth and regeneration after spinal cord injury are hampered by the complexity of the events involved. Here, we present a simple and improved in vitro approach to investigate outgrowth, regeneration of the corticospinal tract, and intrinsic parenchymal responses. We prepared organotypic co-cultures using explants from the motor cortex of postnatal donor mice ubiquitously expressing green fluorescent protein and cervical spinal cord from wild type pups of the same age. Our data show that: a) motor-cortical outgrowth is already detectable after 1 d in culture and is source specific; b) treatment with neurotrophin-3 and C3 transferase from Clostridium botulinum significantly enhances axonal outgrowth during the course of cultivation; c) outgrowing axons form synaptic connections, as demonstrated by immunohistochemistry and calcium imaging; and d) migrating cells of motor-cortical origin can be reliably identified without previous tracing and are mostly neural precursors that survive and mature in the spinal cord parenchyma. Thus, our model is suitable for screening for candidate substances that enhance outgrowth and regeneration of the corticospinal tract and for studying the role of endogenous neural precursors after lesion induction.

A botulinum neurotoxin-like function of Potentilla chinensis extract that inhibits neuronal SNARE complex formation, membrane fusion, neuroexocytosis, and muscle contraction.

CONTEXT: Botulinum neurotoxins (BoNTs) are popularly used to treat various diseases and for cosmetic purposes. They act by blocking neurotransmission through specific cleavage of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Recently, several polyphenols were shown to interfere with SNARE complex formation by wedging into the hydrophobic core interface, thereby leading to reduced neuroexocytosis. OBJECTIVE: In order to find industrially-viable plant extract that functions like BoNT, 71 methanol extracts of flowers were screened and BoNT-like activity of selected extract was evaluated. MATERIALS AND METHODS: After evaluating the inhibitory effect of 71 flower methanol extracts on SNARE complex formation, seven candidates were selected and they were subjected to SNARE-driven membrane fusion assay. Neurotransmitter release from neuronal PC12 cells and SNARE complex formation inside the cell was also evaluated. Finally, the effect of one selected extract on muscle contraction and digit abduction score was determined. RESULTS: The extract of Potentilla chinensis Ser. (Rosaceae)(Chinese cinquefoil) flower inhibited neurotransmitter release from neuronal PC12 cells by approximately 90% at a concentration of 10 µg/mL. The extract inhibited neuroexocytosis by interfering with SNARE complex formation inside cells. It reduced muscle contraction of phrenic nerve-hemidiaphragm by approximately 70% in 60 min, which is comparable to the action of the Ca²âº-channel blocker verapamil and BoNT type A. DISCUSSION AND CONCLUSION: While BoNT blocks neuroexocytosis by cleaving SNARE proteins, the Potentilla chinensis extract exhibited the same activity by inhibiting SNARE complex formation. The extract paralyzed muscle as efficiently as BoNT, suggesting the potential versatility in cosmetics and therapeutics.

Botulinum toxin treatment of myofascial pain: a critical review of the literature.

This is a review of literature relevant to the treatment of myofascial pain syndrome by botulinum injections. The objective is to critically review the studies to see if they are appropriately designed, conducted, and interpreted to provide guidance in the management of myofascial pain. The intent is to better understand the mixed results that these studies have provided. A search was made utilizing PubMed for literature relevant to the use of botulinum toxin in the treatment of myofascial pain. All identifiable series were reviewed, including open label, single-blinded and double-blinded studies, randomized and controlled, or not. In general, small case series of only a few patients were not included unless they made a relevant point and there were no available randomized studies or larger studies. Single case reports were not included. This is not a meta-analysis. The studies were evaluated according to their design and the selection of outcome measurements, and the interpretation of results. The studies were individually critiqued, and an overall assessment and commentary was made of the studies in the field as a whole. Problems that were common to the studies were robust placebo responders, incomplete treatment of a regional myofascial pain syndrome, inappropriate or confounding control populations or treatments, and inappropriate time periods for assessment of outcomes, or misinterpretation of the time-frame of action of botulinum toxin. The studies of the effect of botulinum toxin treatment of myofascial trigger points have had mixed results. However, few studies have been designed to avoid many of the pitfalls associated with a trial of botulinum toxin treatment of trigger points. Better-designed studies may give results that can be used to guide practice based on reliable evidence. At the present time, one must conclude that the available evidence is insufficient to guide clinical practice.

Botulinum toxin injection in epicardial autonomic ganglia temporarily suppresses vagally mediated atrial fibrillation.

BACKGROUND: Autonomic denervation may suppress atrial fibrillation (AF) vulnerability. This study was designed to assess the short- to mid-term effects of botulinum toxin, a cholinergic neurotransmission blocker, on AF inducibility. METHODS AND RESULTS: A total of 23 mongrel dogs were studied. The sinus node and atrioventricular node epicardial fat pads were exposed through a right lateral thoracotomy. Botulinum toxin (Botox, 50 U per fat pad) or 0.9% normal saline (control) was injected into the center of each of the 2 fat pads. The electrophysiological effects were evaluated at 1, 2, and 3 weeks (7 to 8 animals at each time point) with and without cervical vagal stimulation. The vagal stimulation effects on the sinus and atrioventricular nodes were inhibited, and dispersion of atrial effective refractory period was lower at 1 week in the Botox group. Significant suppression of AF inducibility was observed at 1 week but disappeared at 2 and 3 weeks. These changes were not observed in the control group. CONCLUSIONS: Temporary suppression of vagally mediated AF, for at least 1 week, was achieved with botulinum toxin injection in this canine model. This effect might be associated with reduced dispersion of effective refractory period. A temporary autonomic block using botulinum toxin might be a novel therapeutic option for several clinical conditions such as post-cardiac surgery AF.

Botulinum toxin injection in dysphagia syndromes with preserved esophageal peristalsis and incomplete lower esophageal sphincter relaxation.

BACKGROUND: Botulinum toxin injection into the lower esophageal sphincter (LES) treats dysphagia syndromes with preserved peristalsis and incomplete LES relaxation (LESR). We evaluated clinical and esophageal motor characteristics predicting response, and compared duration of efficacy to similarly treated achalasia patients. METHODS: Thirty-six subjects (59 ± 2.2 years, 19F/17M) with incomplete LESR on high resolution manometry (HRM) treated with botulinum toxin injection were identified. Individual and composite symptom indices were calculated, and HRM characteristics extracted. Symptom resolution for 6 months was a primary outcome measure, and repeat botulinum toxin injection, dysphagia recurrence or employment of alternate therapeutic approaches were secondary outcome measures. Duration of response was compared using Kaplan-Meier survival curves to a historical cohort of similarly treated achalasia subjects. KEY RESULTS: Response lasted a mean of 12.8 ± 2.3 months. Symptom relief for >6 months was seen in 58.3%; short (<6 months) response was associated with younger age, higher chest pain index, and esophageal body spastic features (P ≤ 0.04). On multivariate logistic regression, chest pain, younger age and contraction amplitudes >180 mmHg independently predicted <6 months relief (P < 0.05 for each). On survival analysis, relief with a single injection extended to 1 year in 54.8% and 1.5 years in 49.8%, statistically equivalent to that reported by 42 similarly treated achalasia subjects (59 ± 3.2 years, 24F/18M). Symptom relief was more prolonged compared to achalasia when repeat injections were performed on demand (P = 0.003). CONCLUSIONS & INFERENCES: Botulinum toxin injections can provide lasting symptom relief in dysphagia syndromes with incomplete LESR. Prominent perceptive symptoms and non-specific spastic features may predict shorter relief.

Spasticity: revisiting the role and the individual value of several pharmacological treatments.

Though in the last few decades only a few new drugs have come available for the treatment of spasticity, new insights may revise the role and individual value of several pharmacological treatments. Diazepam, baclofen and tizanidine are the most prescribed drugs for the treatment of spasticity. Intrathecal baclofen and local infiltration of botulin toxin are added values in selective patients. Gabapentin is a novelty, and the working mechanism of cannabis has been elucidated. Dantrolene sodium appears to owe its selectivity from the recently discovered ryanodine receptor, with a peripheral effect in muscles. In this review the pathophysiology and epidemiology of spasticity, pharmacology, clinical efficacy and unwanted effects of the different drugs for spasticity are updated.

Botulinum toxin and the facial feedback hypothesis: can looking better make you feel happier?

The facial feedback hypothesis suggests that muscular manipulations which result in more positive facial expressions may lead to more positive emotional states in affected individuals. In this essay, we hypothesize that the injection of botulinum toxin for upper face dynamic creases might induce positive emotional states by reducing the ability to frown and create other negative facial expressions. The use of botulinum toxin to pharmacologically alter upper face muscular expressiveness may curtail the appearance of negative emotions, most notably anger, but also fear and sadness. This occurs via the relaxation of the corrugator supercilii and the procerus, which are responsible for brow furrowing, and to a lesser extent, because of the relaxation of the frontalis. Concurrently, botulinum toxin may dampen some positive expressions like the true smile, which requires activity of the orbicularis oculi, a muscle also relaxed after toxin injections. On balance, the evidence suggests that botulinum toxin injections for upper face dynamic creases may reduce negative facial expressions more than they reduce positive facial expressions. Based on the facial feedback hypothesis, this net change in facial expression may potentially have the secondary effect of reducing the internal experience of negative emotions, thus making patients feel less angry, sad, and fearful.

Development of a cell transducible RhoA inhibitor TAT-C3 transferase and its encapsulation in biocompatible microspheres to promote survival and enhance regeneration of severed neurons.

PURPOSE: Neurons in post-traumatized mammalian central nervous system show only limited degree of regeneration, which can be attributed to the presence of neurite outgrowth inhibitors in damaged myelin and glial scar, and to the apoptosis of severed central neurons and glial cells during secondary Wallerian degeneration. RhoA GTPase has been implicated as the common denominator in these counter-regeneration events, which shows significant and persistent up-regulation for weeks in injured spinal cord and cerebral infarct after stroke. While the exoenzyme C3 transferase is a potent RhoA inhibitor, its extremely low efficiency of cell entry and degradation in vivo has restricted the therapeutic value. This study aims to circumvent these problems by developing a membrane-permeating form of C3 transferase and a biopolymer-based microsphere depot system for sustainable controlled release of the protein. MATERIALS AND METHODS: A membrane-permeating form of C3 transferase was developed by fusing a Tat (trans-activating transcription factor) transduction domain of human immunodeficiency virus to its amino terminal using standard molecular cloning techniques. After confirming efficient cell entry into epithelial and neuroblastoma cells, the resulting recombinant protein TAT-C3 was encapsulated in biocompatible polymer poly(D,L -lactide-co-glycolide) in the form of microspheres by a water-in-oil-in-water (W/O/W) emulsion method. By blending capped and uncapped form of the polymer at different ratios, TAT-C3 protein release profile was modified to suit the expression pattern of endogenous RhoA during CNS injuries. Bioactivity of TAT-C3 released from microspheres was assessed by RhoA ribosylation assay. RESULTS: In contrast to wild-type C3 transferase, the modified TAT-C3 protein was found to efficiently enter NIH3T3 and N1E-115 neuroblastoma cells as early as 6 hours of incubation. The fusion of TAT sequence to C3 transferase imposed no appreciable effects on its biological activity in promoting neurite outgrowth through RhoA inhibition. Characterization of TAT-C3 encapsulation in various blends of capped/uncapped PLGA polymer revealed the 30:70 formulation to be optimal in attaining a mild initial burst release of 25%, followed by a subsequent average daily release of 2.3% of encapsulated protein over one month, matching the change in RhoA level in severed brain and spinal cord. Importantly, TAT-C3 released from the microspheres remained active up to the first three weeks of incubation. CONCLUSION: Enhanced cell entry of TAT-C3 circumvents the need to administer high dose of the protein to site of injury. The encapsulation of TAT-C3 in different blends of capped/uncapped PLGA microspheres allows adjustment of protein release profile to suit the pattern of RhoA expression in injured CNS.

[Potential antinociceptive mechanisms of botulinum toxin]. / Mögliche antinozizeptive Mechanismen von Botulinumtoxin.

Botulinum toxin has been used in pain therapy for several years. Its application in migraine and headaches is particularly interesting. Clinical results have not yet been definitely conclusive, and a uniform model of the mode of action has not been established either. Apart from a purely muscular effect, a direct antinociceptive effect of botulinum toxin has been found in patients, in the preclinical model, and in a clinical pain model. This is contradicted by negative observations in the clinical model of pain, which might be related to methodological deficits. Further basics need to be worked out before arriving at any final result. Clinical studies with patients and pain models should then follow. Studying botulinum toxin within the context of pain will also provide many new insights into pain therapy in general. In which pain model botulinum toxin may play a role in the future, has to be awaited.

The use of botulinum toxin in physical medicine and rehabilitation.

A review of the history and pharmacology of the botulinum neurotoxins is presented. Established mechanisms of action are discussed as well as preliminary evidence of other potential mechanisms, as related to botulinum toxins' antinociceptive properties. Methods of administration, including reconstitution, dilution, and basic injection techniques/principles are reviewed. Safety concerns are also addressed. Various applications relevant to the field of Physical Medicine & Rehabilitation are reviewed, specifically uses in the management of muscle over activity syndromes such as upper motor neuron-related spasticity, dystonias, and painful syndromes including Myofascial Pain Syndromes and headaches. Relevant literature related to these applications is reviewed and discussed. Botulinum toxin therapeutic efficacy and possible reasons for treatment failure (including development of antibody-mediated resistance) are discussed.