Intramuscular Toxin Injection Research Articles

Botulinum neurotoxin type A-cleaved SNAP25 is confined to primary motor neurons and localized on the plasma membrane following intramuscular toxin injection

The mechanism of action of botulinum neurotoxin type A (BoNT/A) is well characterized, but some published evidence suggests the potential for neuronal retrograde transport and cell-to-cell transfer (transcytosis) under certain experimental conditions. The present study evaluated the potential for these processes using a highly selective antibody for the BoNT/A-cleaved substrate (SNAP25197) combined with 3-dimensional imaging. SNAP25197 was characterized in a rat motor neuron (MN) pathway following toxin intramuscular injections at various doses to determine whether SNAP25197 is confined to MNs or also found in neighboring cells or nerve fibers within spinal cord (SC). Results demonstrated that SNAP25197 immuno-reactive staining was colocalized with biomarkers for MNs, but not with markers for neighboring neurons, nerve fibers or glial cells. Additionally, a high dose of BoNT/A, but not a lower dose, resulted in sporadic SNAP25197 signal in distal muscles and associated SC regions without evidence for transcytosis, suggesting that the staining was due to systemic spread of the toxin. Despite this spread, functional effects were not detected in the distal muscles. Therefore, under the present experimental conditions, our results suggest that BoNT/A is confined to MNs and any evidence of distal activity is due to limited systemic spread of the toxin at higher doses and not through transcytosis within SC. Lastly, at higher doses of BoNT/A, SNAP25197 was expressed throughout MNs and colocalized with synaptic markers on the plasma membrane at 6 days post-treatment. These data support previous studies suggesting that SNAP25197 may be incorporated into SNARE-protein complexes within the affected MNs.

Botulinum neurotoxin type A–Cleaved SNAP25 is confined to primary motor neurons and expressed on the plasma membrane following intramuscular toxin injection

Botulinum neurotoxin type A–Cleaved SNAP25 is confined to primary motor neurons and expressed on the plasma membrane following intramuscular toxin injection

Effect of ammodytin L from the venom of Vipera ammodytes on xenopus laevis differentiated muscle fibres and regenerating limbs

Ammodytin L is a non-catalytic, phospholipase-like snake venom toxin from Vipera ammodytes, which shows a cytotoxic activity on differentiated myotubes when tested in vitro. In the range of concentrations in which ammodytin L induced necrosis of myogenic cells in culture, other cell types (erythrocytes, platelets, fibroblasts) did not appear to be affected. To test the in vivo toxicity and the effective cytolytic specificity of ammodytin L we have followed the morphological changes in muscle tissue of Xenopus laevis limbs after intramuscular toxin injection. Only muscular cells were affected by ammodytin L, and the toxin did not induce any morphological change in other cell types. Further evidence of the muscle-specific action of the toxin was obtained from experiments carried out using the Xenopus kidney cell line B3.2 in culture. Ammodytin L was unable to affect parameters of cell viability such as lactate dehydrogenase leakage, [ 3H]thymidine incorporation, growth curves and morphological changes. Moreover, direct ammodytin L application to cultured regenerative limbs did not provoke alterations in undifferentiated myoblasts. These data suggest that ammodytin L, like other phospholipase-like toxins, exerts its toxicity by selectively damaging differentiated muscle fibres.

Miniature end-plate potentials at mammalian neuromuscular junctions poisoned by botulinum toxin.

IT is generally accepted that botulinum toxin entirely blocks transmitter release from motor nerve terminals without affecting nerve conduction or the sensitivity of the muscle membrane to acetylcholine. In particular, it has been reported that with both acute and chronic intoxication with type A botulinum, miniature end-plate potentials (m.e.p.p.s.) disappear completely from a muscle at about the time that transmission is blocked1,2. The action of botulinum toxin has been reinvestigated following acute application of toxin to the rat diaphragm in vitro, and chronic paralysis of rat soleus muscle following a single intramuscular injection of toxin; miniature potentials have been observed to persist following blockade of neuromuscular transmission.

The relationship of toxin and antitoxin injection site to tetanus development in the rat.

The pattern of development of symptoms was studied following injection of 3 MLD (minimum lethal dose) of tetanus toxin in each of the following sites in the rat: vein, muscle, endoneurium, epineurium, spinal cord, subarachnoid space, and subdural space. Similar observations were made when 3 MPD (minimum protective dose) of antitoxin were injected into each of the above sites a few minutes after the intramuscular injection of toxin. Local tetanus followed intramuscular, endoneurial, and epineurial injection of the toxin; blood-borne tetanus also appeared in the latter instance. Tetanus dolorosus followed intraspinal injection of toxin. Both dorsal tetanus and blood-borne tetanus developed after subdural administration of toxin. Blood-borne tetanus, alone, appeared following intravenous and subarachnoid injection of toxin. In no case did injection of antitoxin at the various sites listed above prevent local tetanus from developing after intramuscular injection of toxin. Of the various sites injected with antitoxin, the intramuscular, intraspinal, and intravenous were the most effective. It is suggested that the endoneurial tissue spaces serve as a conduit for tetanus toxin from the muscle to the CNS. The perilemma of the peripheral nerve trunk may act as a selectively permeable membrane which permits tetanus toxin to pass from the epineurium to the endoneurium. Outward diffusion of toxin from the endoneurial spaces is apparently markedly reduced. The antitoxin probably is prevented from permeating this barrier in either direction. The importance of absorption of tetanus toxin by the lymphatic and blood circulation is discussed.

Investigations on the Pathogenesis of Tetanus III

Summary and Conclusions In this and previous communications, investigations on the pathogenesis of tetanus have been reported. The theories of Courmont and Doyon, Meyer and Ransom and Abel have been subjected to experimental tests. We believe to have established the following facts: Tetanal toxin does not reach the C-N-S directly by way of the circulation. This follows from an observation referred to as the R-(route) phenomenon. It was found that the amount of circulating antitoxin which protects animals from death is determined by the route of introduction of the toxin. The same test-dose of toxin, when injected by the intracutaneous or intramuscular routes, requires up to 80 times more antitoxin than after intravenous injection. This result is obviously incompatible with the assumption that the toxin reaches the C-N-S from the tissue after absorption into the circulation.Tetanus cannot be explained by an action of the toxin on the sensory nerve-endings (Courmont and Doyon). Section of the posterior spinal nerve-roots does not influence the R-phenomenon or local tetanus.Tetanus cannot be explained by the activity of a secondary toxic substance formed in the muscle (Courmont and Doyon) or in the C-N-S (Firor, Lamont and Shumacker). Transverse lesions of the dorsal cord, followed by intravenous injection of antitoxin and intramuscular injection of toxin into one hind leg, confines tetanus to the lower extremities and considerably prolongs the survival-time of the experimental animals. This shows that generalized tetanus cannot be explained by a vascular transport of a secondary toxic substance to higher levels of the C-N-S.Tetanal toxin reaches the C-N-S by way of the motor nerves. The R-phenomenon disappears after section of the peripheral nerves or anterior roots.Tetanal toxin spreads within the C-N-S. As mentioned under the third conclusion, after intravenous injection of antitoxin, intramuscular injection of toxin into one hind leg and transverse lesion of the cord, tetanus remains confined to the lower extremities. This shows that interruption of the neural pathways within the cord prevents the passage of the toxin to higher levels of the C-N-S. Moreover, the appearance of tetanus in both hind legs proves the migration of the toxin from one side of the cord to the other.Local tetanus is of central origin. Under optimal experimental conditions antitoxin is 80 times more potent by the intraventricular than the intravenous route in preventing local tetanus in the hind leg. Since the possibility could be excluded that the intraventricularly injected antitoxin reaches the myoneural junction by way of the nerve this experiment shows conclusively that local tetanus is due to an action of the toxin on the C-N-S.Our experiments therefore confirm the nerve-carriage theory of Meyer and Ransom in all essential points.Our results provide conclusive evidence for the existence of the blood-C-N-S barrier. Since tetanal toxin is extremely toxic when injected into the lumbar cord its inability to reach the C-N-S by way of the circulation can be explained only by an impermeability of the capillaries of the C-N-S to the toxin. This, in connection with the electro-negative charge of tetanal toxin, confirms the rule of Friedemann and Elkeles according to which the blood-C-N-S barrier is impermeable to electro-negative substances.It is now established that an inanimate substance can be transmitted within the nerve to the central nervous system. The implications of this fact for the pathogenesis of neurotropic virus diseases are discussed.