targeted-toxins

Q: When performing intraparenchymal injections of immunotoxin, what is the proper volume to use? Is it better to induce two half-portions per hemisphere or is a higher concentration better? At what concentration do you expect necrosis or inflammation?

A: There is no one answer to the question of injection volume. Practically speaking, we have observed that large or extended structures such as the entire cholinergic basal forebrain (CBF) of rats are difficult to ablate with one single injection of 192-Saporin (192-IgG-SAP, Cat. #IT-01). The best results were obtained with 3-5 separate 0.5-1.0 µl injections. For even larger targets such as the CBF in primates, other strategies may be necessary. Oldfield and co-workers have reported success in delivering cytotoxic chemotherapy to large volumes of brain using long, slow infusions of solutions containing a low concentration of toxin (convective delivery). This procedure delivers toxin by bulk fluid flow rather than diffusion and avoids high local toxin concentrations around the infusion catheter or pipette. High local concentrations of toxin may compromise selectivity and produce non-specific cytotoxicity. This can occur when neurons and glia take up toxic amounts of saporin by bulk fluid phase endocytosis, rather than receptor-mediated endocytosis. With direct intraparenchymal injections, local necrosis can occur with surprisingly small doses of toxin. For example, 60 ng of SP-SAP (Cat. #IT-07) or dermorphin-SAP (Cat. #IT-12) into the rat striatum injected in 1 µl typically produces some necrosis in the center of the injection site. With immunotoxins such as 192-Saporin (192-IgG-SAP) or Anti-DBH-SAP (Cat. #IT-03), 200 ng in 0.5 µl may barely produce a trace of local damage.

Q: We are interested in the anti-Thy-1 nephritis model in rats. I want to know the titer of OX7-SAP (Cat. #IT-02) and how much we have to expend for each rat to establish the model?

A: The “titer” of OX7-SAP is rather difficult to define. It is not known precisely how many molecules of this immunotoxin are necessary to kill a thymic-derived (Thy-1-expressing) lymphocyte in vivo. Also, since OX7-SAP kills Thy-1-expressing lymphocytes, it may prove difficult to induce Thy-1 nephritis with the immunotoxin. In humans, proteinuria was reported in clinical trials using immunotoxins for treatment of cancer, but we do not know of any comparable data in rats. Probably the only way to determine the appropriate dose would be a dose ranging study.

Q: I have been doing research with 192-Saporin (192-IgG-SAP, Cat. #IT-01) for 2-3 years now. I have read in the literature that animals with cholinergic lesions often get sick following surgery and require potatoes, apples, lettuce and saline injections. They may even stop eating or drinking all together. I have followed these practices in the past, but stopped when it didn’t seem to make a difference. (I use a very small insignificant dose that is not prone to make animals ill). This is the first death I have had even remotely possibly related to the toxin. In short, the animal lost 64 grams over a period of 2 weeks, and expired 1-2 days thereafter. I weighed her at death and she was 139 grams (91 gram difference from her initial surgery weight). The rats in our colony are fed and given water ad libitum. However, we think that she dehydrated. She was given the same dose (1.1 microliters) as all of the other rats in the experiment. I do have other rats that were given injections from the same lot that do not appear to be sick or losing weight. I’m not sure what you can do with this information, but I would be grateful for whatever help you can offer.

In large series of intraventricular injections of 192-Saporin (192-IgG-SAP), I have never encountered quite the same sequence of events you describe. Death after intracranial toxin injection can reflect several possible misadventures including but not limited to:

1. Contamination of toxin solution with endotoxin resulting in death without awakening from anesthesia (usually due to bacterial contamination from prolonged exposure of toxin solution to room temperature),

2. Fatal intracranial hemorrhage which may result in delayed death depending on location and volume of bleeding,

3. Intracranial abscess (extremely unusual) from injection of contaminated solution, or

4. Unrelated bacterial, viral or parasitic systemic disease.

Q: Another one of my 192-Saporin-treated (or 192-IgG-SAP, Cat. #IT-01) rats is having an adverse reaction, including paralysis of the lower extremities. She has lost 40 grams in the past 3-4 days. Could this be Purkinje cell damage; does that happen after five weeks?

A: Purkinje cell damage after intraventricular injection of 192-Saporin (192-IgG-SAP) typically is manifest not by hind limb paralysis but rather tremor (shaking) and ataxia (clumsiness, poor balance). At less than lethal doses, 192-Saporin (192-IgG-SAP) does not produce paraparesis. Something else is going on. Rats develop hind limb paralysis from a variety of toxic or metabolic systemic insults in addition to specific nervous system disorders. The presence of rapid weight loss suggests the rat is systemically ill rather than an effect of a sub-lethal dose of 192-Saporin (192-IgG-SAP).

Selected references on convective delivery of toxin to brain:

Nguyen TT, Pannu YS, Sung C, Dedrick RL, Walbridge S, Brechbiel MW, Garmestani K, Beitzel M, Yordanov AT, Oldfield EH (2003) Convective distribution of macromolecules in the primate brain demonstrated using computerized tomography and magnetic resonance imaging. J Neurosurg 98(3):584-590.

Morrison PF, Chen MY, Chadwick RS, Lonser RR, Oldfield EH (1999) Focal delivery during direct infusion to brain: role of flow rate, catheter diameter, and tissue mechanics. Am J Physiol 277(4 Pt 2):R1218-R1229.

Lonser RR, Corthesy ME, Morrison PF, Gogate N, Oldfield EH (1999) Convection-enhanced selective excitotoxic ablation of the neurons of the globus pallidus internus for treatment of Parkinsonism in nonhuman primates. J Neurosurg 91(2):294-302.

Wood JD, Lonser RR, Gogate N, Morrison PF, Oldfield EH (1999) Convective delivery of macromolecules into the naive and traumatized spinal cords of rats. J Neurosurg 90(1 Suppl):115-120.

Lonser RR, Gogate N, Morrison PF, Wood JD, Oldfield EH (1998) Direct convective delivery of macromolecules to the spinal cord. J Neurosurg 89(4):616-622.

Laske DW, Morrison PF, Lieberman DM, Corthesy ME, Reynolds JC, Stewart-Henney PA, Koong SS, Cummins A, Paik CH, Oldfield EH (1997) Chronic interstitial infusion of protein to primate brain: determination of drug distribution and clearance with single-photon emission computerized tomography imaging. J Neurosurg 87(4):586-594.

Lieberman DM, Laske DW, Morrison PF, Bankiewicz KS, Oldfield EH (1995) Convection-enhanced distribution of large molecules in gray matter during interstitial drug infusion. J Neurosurg 82(6):1021-1029.

Bobo RH, Laske DW, Akbasak A, Morrison PF, Dedrick RL, Oldfield EH (1994) Convection-enhanced delivery of macromolecules in the brain. Proc Natl Acad Sci U S A 91(6):2076-2080.

Morrison PF, Laske DW, Bobo H, Oldfield EH, Dedrick RL (1994) High-flow microinfusion: tissue penetration and pharmacodynamics.Am J Physiol 266(1 Pt 2):R292-305.

See: Targeted Toxins

Lappi DA (2003) Featured Article: A new immunotoxin for targeting dopaminergic neurons. Targeting Trends 4(3)

Related Products: Anti-DAT-SAP (Cat. #IT-25)

Read the featured article in Targeting Trends.

Vierck CJ, Kline RH, Wiley RG (2003) Intrathecal substance p-saporin attenuates operant escape from nociceptive thermal stimuli. Neuroscience 119(1):223-232. doi: 10.1016/s0306-4522(03)00125-8

Summary: Administration of SP-SAP (Cat. #IT-07) eliminates sensitization of nocifensive reflexes. The authors investigate whether SP-SAP elimination of neurokinin-1 receptor-expressing neurons in the lumbar spinal cord affects nociceptive sensitivity in general, or preferentially affects nociception dependent on spinal and brainstem, or cerebral processing. Rats treated with spinal intrathecal injections of 175 ng of SP-SAP showed attenuated thermal hyperalgesia, and no secondary hyperalgesia, while innate reflexes were unaffected by SP-SAP treatment.

Related Products: SP-SAP (Cat. #IT-07)

Ricceri L (2003) Behavioral patterns under cholinergic control during development: lessons learned from the selective immunotoxin 192 IgG saporin. Neurosci Biobehav Rev 27(4):377-384. doi: 10.1016/s0149-7634(03)00068-x

Summary: The author reviews the effects of 192-Saporin (Cat. #IT-01) neonatal lesions (0.42 µg in each hemisphere) on the cholinergic basal forebrain system in rats. Short-term effects are seen in pups in learning tasks, as well as ultrasound vocalizations. Longer term effects are seen in task-specific behaviors. Data suggest that the extent of these effects are linked to the attentional load of the task. The age of the animal when lesioned may also play a role in the extent of the deficits caused by 192-Saporin; studies show that early in the first week of life is a particularly vulnerable period.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Pappas BA, Sherren N (2003) Neonatal 192 IgG-saporin lesion of forebrain cholinergic neurons: focus on the life span?. Neurosci Biobehav Rev 27(4):365-376. doi: 10.1016/s0149-7634(03)00067-8

Summary: In this review, the authors discuss the use of 192-Saporin in the investigation of neurodevelopmental disorders, and propose that the effects of these lesions are amplified as the animal ages and experiences normal age-related synapse loss.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Kanai T, Uraushihara K, Okazawa A, Hibi T, Watanabe M (2003) Macrophage-derived IL-18 targeting for the treatment of Crohn’s disease. Curr Drug Targets Inflamm Allergy 2(2):131-136. doi: 10.2174/1568010033484250

Summary: A single intravenous injection of Mac-1-SAP (Cat. #IT-06) significantly reduced the amount of intestinal inflammation in a 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis model.

Related Products: Mac-1-SAP mouse/human (Cat. #IT-06)

Conner JM, Culberson A, Packowski C, Chiba AA, Tuszynski MH (2003) Lesions of the basal forebrain cholinergic system impair task acquisition and abolish cortical plasticity associated with motor skill learning. Neuron 38(5):819-829. doi: 10.1016/s0896-6273(03)00288-5

Summary: Neuronal plasticity has been associated with normal learning. The authors wished to investigate the role of the cholinergic basal forebrain (CBF) system in learning motor skills. Rats received bilateral 95-ng injections of 192-Saporin (Cat. #IT-01) in either the medial septum, the nucleus basalis magnocellularis, or both. The results indicate that lesioned animals, with many aspects of attention still preserved, are unable to adapt attention to meet the demands of a particular task. The authors conclude that the CBF system may be implicated in learning forms that require plasticity of cortical representations.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Rudick CN, Gibbs RB, Woolley CS (2003) A role for the basal forebrain cholinergic system in estrogen-induced disinhibition of hippocampal pyramidal cells. J Neurosci 23(11):4479-4490. doi: 10.1523/JNEUROSCI.23-11-04479.2003

Summary: Estrogen plays a strong regulatory role in control of synaptic input to the hippocampus of female rats. Injection of 0.22 µg of 192-Saporin (Cat. #IT-01) directly into the medial septum eliminated NGFr-positive cholinergic neurons of the basal forebrain, producing evidence that estrogen-induced disinhibition is partially dependent on these neurons. GABAergic synapses were also found to be involved in this system.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Nakatsuka T, Tsuzuki K, Ling JX, Sonobe H, Gu JG (2003) Distinct roles of P2X receptors in modulating glutamate release at different primary sensory synapses in rat spinal cord. J Neurophysiol 89(6):3243-3252. doi: 10.1152/jn.01172.2002

Summary: P2X receptors are important modulating neurons in the spinal cord. These authors used IB4-SAP (Cat. #IT-10) to target a neuronal subset, those neurons expressing P2X3 receptors. 2 µg of IB4-SAP were injected directly into the sciatic nerve on one side. Histological examination showed efficient removal of IB4 and P2X3-staining ipsilaterally in the dorsal horn outer laminae. Behavioral experiments showed intact modulation of glutamate release in the absence of P2X3-positive neurons, indicating involvement by other P2X neurons.

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Calza L, Giuliani A, Fernandez M, Pirondi S, D’Intino G, Aloe L, Giardino L (2003) Neural stem cells and cholinergic neurons: Regulation by immunolesion and treatment with mitogens, retinoic acid, and nerve growth factor. Proc Natl Acad Sci U S A 100(12):7325-7330. doi: 10.1073/pnas.1132092100

Summary: The authors explore the influence of exogenous administration of hormones, cytokines, and neurotrophins on stem cells following a lesion. Rats were treated with 2 or 3 µg of 192-Saporin (Cat. #IT-01) into the cerebral ventricles, which induced a lesion of the cholinergic system in the basal forebrain. The surgery was followed by infusion of EGF, bFGF, and NGF into the lesioned area, as well as addition of retinoic acid to the food pellets. This pharmacological control of endogenous neural stem cells increased the number of proliferating cells in both lesioned and non-lesioned animals, as well as improved performance in a water maze test.

Related Products: 192-IgG-SAP (Cat. #IT-01)