Q: Our lab is getting ready to begin a project using one of your targeted toxins. We already did a preliminary experiment to try out the material, but we have a couple of questions before we start the larger project. First, do you have any protocols or references for injecting intrathecally?
A: Thank you for your inquiry. We appreciate the opportunity to get involved in projects before they begin. At Advanced Targeting Systems, we do not do any in vivo work, just in vitro, however we have collaborated with many fine laboratories that have good experience with intrathecal injections. If you search PubMed with the keywords ‘saporin’ and ‘intrathecal’ you will be able to view references that will give you good information on techniques and protocols. Prior to beginning your project you will want to submit your animal care guidelines to your IACUC committee. Turner et al. published an article that will be helpful regarding intrathecal injections. [1]
Q: The second question is in two parts: 1) how do we determine the appropriate dose, and 2) how do we know saporin is not killing indiscriminately at that dose?
A: You should always use a control when determining the appropriate dose. A basic premise of the ATS targeting technology is that if a control (saporin alone or a control conjugate) evokes a response, then the dose is too high. Whenever a new shipment of targeted toxin is received, the proper working dilution should be ascertained before beginning a project. The targeted toxin data sheet states:
“There may be lot-to-lot variation in material; working dilutions must be determined by end user. If this is a new lot, assess the proper working dilution before beginning a full experimental protocol.”
If you search on the ATS website for the species and route of administration you plan to use, you can look through the publication summaries and see the dose that was used for that particular study. That will give you a ballpark range in which to start your dose titration. Just keep in mind: if the control kills cells, the dose is too high.
Liu H, Yan W, Lu X, Zhang X, Wei J, Wang X, Wang T, Wu T, Cao J, Shao C, Zhou F, Zhang H, Zhang P, Zang T, Lu X, Cao J, Ding H, Zhang L (2014) Role of the cerebrospinal fluid-contacting nucleus in the descending inhibition of spinal pain transmission. Exp Neurol 261:475-485. doi: 10.1016/j.expneurol.2014.07.018
Summary: The first synapse in the pain pathway is in the spinal dorsal horn, and several sites are involved in the descending control of pain. Previous studies have suggested that cerebrospinal fluid-contacting neurons may facilitate signal transmission and substance transport between the brain parenchyma and the CSF, including processes that modulate pain transmission. The authors administered CTB-SAP (Cat. #IT-14) into the right lateral ventricle of rats. Saporin (Cat. #PR-01) was used as a control. The results indicate that the 5-HT pathway contacting the CSF is an important piece in the descending inhibitory system controlling spinal transmission of pain.
Flak J, Myers B, Solomon M, McKlveen J, Krause E, Herman J (2014) Role of paraventricular nucleus-projecting norepinephrine/epinephrine neurons in acute and chronic stress. Eur J Neurosci 39:1903-1911. doi: 10.1111/ejn.12587
Summary: Chronic stress can cause dysregulation of the paraventricular nucleus (PVN) of the hypothalamus, resulting in structural and function changes in the neurons involved. There are data indicating that post-stress enhancement of norepinephrine is involved in the processing of chronic stress. In this work the authors investigated the hypothesis that PVN-projecting norepinephrine/epinephrine (NE/E) neurons are necessary for chronic stress-induced drive of the hypothalamic-pituitary-adrenocortical (HPA) axis. Rats received bilateral 8.82 ng injections of anti-DBH-SAP (Cat. #IT-03) into the PVN. Saporin (Cat. #PR-01) was used as a control. Lesioned animals displayed attenuated peak ACTH, indicating that NE/E neurons are required for ACTH release in the HPA axis during chronic stress.
Li AJ, Wang Q, Dinh TT, Powers BR, Ritter S (2014) Stimulation of feeding by three different glucose-sensing mechanisms requires hindbrain catecholamine neurons. Am J Physiol Regul Integr Comp Physiol 306(4):R257-R264. doi: 10.1152/ajpregu.00451.2013
Summary: The glucoregulatory system of the brain requires catecholamine neurons in the hindbrain. he sensory mechanisms and connected circuitry controlling the response to glucose deficit are not well understood. In order to investigate different drugs that stimulate food intake but interfere with cellular glucose metabolism and transport the authors administered 82 ng of anti-DBH-SAP (Cat. #IT-03) into the paraventricular nucleus as bilateral injections. Saporin (Cat. #PR-01) was used as a control. Lesioned animals did not increase food intake in response to any of the drugs, indicating that stimulation of food intake is activated through a catecholamine-dependent pathway.
Ye Y, Bae S, Viet CT, Troob S, Bernabe D, Schmidt BL (2014) IB4(+) and TRPV1(+) sensory neurons mediate pain but not proliferation in a mouse model of squamous cell carcinoma. Behav Brain Funct 10(1):5. doi: 10.1186/1744-9081-10-5
Objective: To evaluate subtypes of sensory neurons involved in cancer pain and proliferation.
Summary: IB4(+) neurons play an important role in cancer-induced mechanical allodynia, while TRPV1 mediates cancer-induced thermal hyperalgesia. Characterization of the sensory fiber subtypes responsible for cancer pain could lead to the development of targeted therapeutics.
Usage: IB4(+) neurons play an important role in cancer-induced mechanical allodynia, while TRPV1 mediates cancer-induced thermal hyperalgesia. Characterization of the sensory fiber subtypes responsible for cancer pain could lead to the development of targeted therapeutics.
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: The figure below illustrates the time course of cell death very effectively. Internalization and cytotoxicity of SP-SAP in primary cultures of neonatal spinal cord neurons. Confocal image of neurons where the Substance P receptor; NK1R (SPR) immunofluorescence (A, C, D) appears red, areas of concentrated SPR immunofluorescence appear yellow. (A, C, and D) SPR immunofluorescence in neurons 2 hours, 1 day, and 4 days, respectively, after treatment with SP-SAP. (B) Confocal image showing SAP immunofluorescence (yellow) 2 hours after SP-SAP treatment.
These images were projected from 14 optical sections acquired at 0.8-mm intervals with a 603 lens. Bar, 25 mm.
It is recommended that you wait for two weeks to allow for all debris to be cleared and the animal to regain normal eating and sleeping habits.
Li AJ, Wang Q, Dinh TT, Wiater MF, Eskelsen AK, Ritter S (2013) Hindbrain catecholamine neurons control rapid switching of metabolic substrate use during glucoprivation in male rats. Endocrinology 154(12):4570-4579. doi: 10.1210/en.2013-1589
Summary: Previous work has shown that corticosterone secretion in response to glucoprivation is at least in part controlled by hindbrain catecholamine neurons in the paraventricular nucleus of the hypothalamus (PVH). In this work the authors investigate the metabolic consequences of lesioning these neurons. Rats received bilateral 82-ng infusions of Anti-DBH-SAP (Cat. #IT-03) into the PVH. Saporin (Cat. #PR-01) was used as a control. Although lesioned animals had the same energy expenditure and locomotor activity as controls, they also had a higher respiratory exchange ratio, indicating a reduced ability to switch from carbohydrate to fat metabolism in response to glucoprivation.
da Silva EF, Freiria-Oliveira AH, Custodio CH, Ghedini PC, Bataus LA, Colombari E, de Castro CH, Colugnati DB, Rosa DA, Cravo SL, Pedrino GR (2013) A1 noradrenergic neurons lesions reduce natriuresis and hypertensive responses to hypernatremia in rats. PLoS One 8(9):e73187. doi: 10.1371/journal.pone.0073187
Summary: Using bilateral 63-ng injections of Anti-DBH-SAP (Cat. #IT-03) into two levels of the caudal ventrolateral medulla, the authors assessed several pressor responses to infusion of hypertonic saline. Saporin (Cat. #PR-01) was used as a control. The results suggest that medullary noradrenergic A1 neurons are involved in the regulation of some responses to acute changes in body fluid composition.
