FAQ

Q: How long does it take to see the cell death occurring from the use of targeted toxins using saporin? Is there a time course of hours or days?

A: Details of the time course of early events have not been extensively studied. After ricin injections into the cervical vagus nerve, the proximal nerve becomes unresponsive to electrical stimulation between 36 and 48 hours. After septal injection of 192-Saporin (192-IgG-SAP, Cat. #IT-01), hippocampal theta rhythm begins to diminish on the third postoperative day and reaches a minimum by 7 days which is maintained indefinitely. Anatomical disintegration is complete within 10-14 days after injection of most toxins.

Q: Will this time course be the same regardless of the targeted toxin used or the method of administration?

A: Presumably, injection of toxin into the vicinity of target cell bodies and dendrites should produce effects somewhat sooner than toxin injections into axonal terminal fields where retrograde axonal transport must first deliver toxin to the perikarya. In the cervical vagus, based on transport times for ricin and inhibition of toxin transport by vincristine, we concluded that fast axonal transport is involved. Colchicine coinjected intraventricularly with 192-Saporin (192-IgG-SAP, Cat. #IT-01) prevents destruction of cholinergic basal forebrain neurons suggesting that fast axonal transport also is involved with i.c.v. toxin injections. Consequently, the delay introduced by injecting toxin into axon terminal fields is usually a few hours at most.

Q: What are some assays/methods to use to be able to graphically demonstrate cell death?

A: Toxin-induced cell death can be observed and documented with a variety of techniques. Often the easiest is simple Nissl staining because all of the RIP toxins (ricin, volkensin, saporin) produce profound chromatolysis that is readily apparent in Nissl stains (i.e. cresyl violet).

Electron microscopy can demonstrate details of neuron degeneration including loss of axon terminals at a distance from the cell body which can be useful in anatomic tracing studies.

Typically, target neurons express proteins that can be visualized with immunocytochemical techniques. Thus, immunofluorescence or peroxidase immunohistochemistry can be useful in detecting loss of staining for target molecules and co-expressed molecules in the neurons being targeted. The use of multiple markers is recommended to insure that cell loss occurred rather than down regulation of marker expression.

See: Targeted Toxins

Q: What are the options for delivery of targeted toxins?

A: The options for toxin delivery are varied and limited only by investigator ingenuity. Generally, injection has been the route of choice. Some toxins can be given intravenously, such as 192-Saporin (192-IgG-SAP, Cat. # IT-01) or anti-DBH-SAP (Cat. # IT-03), in which case all cells expressing p75 or dopamine beta-hydroxylase and exposed to the systemic circulation are potential targets. Intravenous injections will not deliver toxins to the CNS.

Subarachnoid injections have been used successfully for immunotoxins and peptide toxins such as SP-SAP (Cat. # IT-07).

Direct intraparenchymal injections have been used to restrict toxin application to just a few target cells. However, intraparenchymal injections require careful attention to injection technique and are impractical for large target structures.

Q: When injecting directly into tissue, are there any special techniques that should be used?

A: Direct injections into brain or spinal cord have been used successfully by some investigators. Specifics of toxin dose, concentration, injection volume and speed of injection have varied considerably. If a high concentration of toxin is deposited locally, lesion specificity is often lost. Presumably, if toxin concentration is too high, cellular uptake by non-specific bulk fluid-phase endocytosis (pinocytosis) can internalize enough saporin to be lethal.

There is currently interest in “convective” delivery techniques developed in the laboratory of Dr. Edward Oldfield at the NIH. The basic principle is to deliver a relatively large concentration slowly over an extended period, often using a rather dilute solution. The parameters for any given species and injection site need to be determined by pilot experiments.

Q: What sort of special care should be given to the animal after administration of the targeted toxin?

A: The toxins generally bind and internalize within minutes, although some immunotoxins circulate for longer periods if injected intravenously. However, no significant amount of active toxin is excreted. So, animals can be returned to group housing immediately after toxin injection. The only special requirements may derive from the specific target being studied. For example, rats given intraventricular 192-Saporin (192-IgG-SAP, Cat. # IT-01) develop decreased fluid and food intake for several days after injection. Since the adipsia is significant, providing the animals with fresh, juicy vegetables, such as cucumber or potatoes, can help.

Rats injected intraventricularly with anti-DBH-SAP (Cat. # IT-03) will lose considerable body weight and are slow to regain. They, too, may benefit from food supplements, including nuts and other high calorie appetizing treats. Otherwise, common sense care of any neurologic deficits is indicated depending on the target and toxin being used.

See: Targeted Toxins

Q: Can you use targeted toxins in vivo?

A: Yes, Molecular Neurosurgery is designed as a tool for in vivo use.

Q: How do you recommend administration of the targeted toxin?

A: There are several ways to administer the toxins depending on the cells being targeted:

1. Direct intraparenchymal pressure microinjection can be used to deliver the targeted toxin directly to target cells. This approach has been used successfully with several toxins, including SP-Saporin (SP-SAP, Cat. #IT-07), in the striatum to kill striatal interneurons that express the NK-1 receptor. Long slow infusions (0.1 µl/min) are probably the best way to do intraparenchymal injections. [1]

2. Targeted toxins can also be injected into terminal fields and retrogradely transported to the cell bodies. This approach has been used successfully to selectively destroy locus coeruleus noradrenergic neurons that project to the olfactory bulb by injecting anti-DBH-saporin (Anti-DBH-SAP, Cat. #IT-03) into the olfactory bulb.[2]

Intracortical injections of 192-Saporin (192-IgG-SAP, Cat. #IT-01) also have been used to destroy cholinergic basal forebrain neurons projecting to the injected patch of cortex.[3]

Lumbar subarachnoid injections of SP-Saporin (SP-SAP, Cat. #IT-07) can destroy lamina I neurons in the dorsal horn that express the NK-1 receptor.[4]

3. Lastly, SP-Saporin (SP-SAP, Cat. #IT-07) has also been applied directly to the surface of the spinal cord to kill lamina I neurons expressing NK-1 receptor. In all cases, pilot studies to determine optimal toxin dose and injection parameters are recommended.

See: Targeted Toxins

References

1. Wiley RG et al. Destruction of neurokinin-1 receptor expressing cells in vitro and in vivo using substance P-saporin. Neurosci Lett 230:97-100, 1997.
2. Blessing WW et al. Destruction of locus coeruleus neuronal perikarya after injection of anti-dopamine-beta-hydroxylase immunotoxin into the olfactory bulb of the rat. Neurosci Lett 243:85-88, 1998.
3. Wiley RG et al. Immunolesioning: Selective destruction of neurons using immunotoxin to rat NGF receptor. Brain Res 562:149-153, 1991.
4. Mantyh PW et al. Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the substance P receptor. Science 278:275-279, 1997.

Q: What is a second immunotoxin?

A: ATS’s second immunotoxins are conjugations of a secondary antibody (as of December 2000, either goat anti-mouse IgG or goat anti-rabbit IgG) to the ribosome-inactivating protein, saporin.

Q: How does a second immunotoxin target?

A: The second immunotoxin uses the secondary antibody to “piggyback” onto your primary antibody in order to evaluate the ability of the primary antibody to internalize.

Q: What happens when the second immunotoxin gets inside the cell?

A: If the second immunotoxin is internalized, saporin will inactivate the ribosomes of the cell, thereby causing cell death.

Q: Are there different types of second immunotoxins available?

A: Yes, please see our catalog listing online for a complete list.

Q: What is the ratio of antibody to second immunotoxin for in vitro testing?

A: Both Mab-ZAP and Rab-ZAP have been shown effective in concentrations ranging from 0.5 to 2 moles of primary antibody per mole of second immunotoxin.

See: ZAP Conjugates (Second Immunotoxins)