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Enhanced evoked excitatory transmitter release in experimental neuropathy requires descending facilitation.
Gardell LR, Vanderah TW, Gardell SE, Wang R, Ossipov MH, Lai J, Porreca F (2003) Enhanced evoked excitatory transmitter release in experimental neuropathy requires descending facilitation. J Neurosci 23(23):8370-8379. doi: 10.1523/JNEUROSCI.23-23-08370.2003
Summary: The authors examine whether afferent discharge produced by nerve injury and central changes in experimental neuropathic pain might interact at the spinal level. Rats were treated with 48 ng of dermorphin-SAP (Cat. #IT-12) and various markers for neuropathic pain were evaluated. The results link several consequences of the post-injury state, including support for increased afferent input as a driving force for neuropathic pain.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
Targeted toxins in pain.
Wiley RG, Lappi DA (2003) Targeted toxins in pain. Adv Drug Deliv Rev 55(8):1043-1054. doi: 10.1016/s0169-409x(03)00102-9
Summary: The authors discuss the use of ‘molecular neurosurgery’ in the study of nociception. Applications using targeted toxins, which include immunotoxins, protein-toxin conjugates, or peptide-toxin conjugates, are illustrated. The authors describe the use of these molecules as research tools, as well as their potential for therapeutics. A helpful table is included that lists neuronal surface markers and class of cells targeted for each targeted toxin. Reagents discussed: CTB-SAP (Cat. #IT-14), IB4-SAP (Cat. #IT-10), OX7-SAP (Cat. #IT-02), 192-Saporin (Cat. #IT-01), ME20.4-SAP (Cat. #IT-15), Anti-DBH-SAP (Cat. #IT-03), Anti-DAT-SAP (Cat. #IT-25), SP-SAP (Cat. #IT-07), Dermorphin-SAP (Cat. #IT-12), Orexin-SAP (Cat. #IT-20), CRF-SAP (Cat. #IT-13), and acetylated LDL-SAP (Cat. #IT-08).
Related Products: CTB-SAP (Cat. #IT-14), IB4-SAP (Cat. #IT-10), OX7-SAP (Cat. #IT-02), 192-IgG-SAP (Cat. #IT-01), ME20.4-SAP (Cat. #IT-15), Anti-DBH-SAP (Cat. #IT-03), Anti-DAT-SAP (Cat. #IT-25), SP-SAP (Cat. #IT-07), Dermorphin-SAP / MOR-SAP (Cat. #IT-12), Orexin-B-SAP (Cat. #IT-20), CRF-SAP (Cat. #IT-13), Acetylated LDL-SAP (Cat. #IT-08)
Dosing, Volume, and Animal Care
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
Retrograde Transport
Q: I’m interested in using SAP to eliminate cells through retrograde transport, like OX7-SAP (Cat. #IT-02) and IB4-SAP (Cat. #IT-10) have been used. Can you explain how retrograde transport works and if it is possible for this to work with dermorphin-SAP (Cat. #IT-12)? What determines whether a targeted toxin will be able to be used in retrograde transport?
A: Current evidence indicates that effective suicide transport agents undergo endocytosis at nerve terminals followed by retrograde axonal transport of the endocytic vesicles containing the toxin. Experiments using vincristine have shown that the retrograde axonal transport of suicide transport toxins utilizes the fast transport system (microtubules). However, it is not known what determines whether or not a specific toxin-ligand undergoes axonal transport after internalization.
Empirically, it has been observed that immunotoxins (OX7-SAP, 192-Saporin or 192-IgG-SAP [Cat. #IT-01], anti-DBH-SAP [Cat. #IT-03]) and lectin-toxins (ricin, volkensin, IB4-SAP) all undergo retrograde axonal transport and are therefore effective suicide transport agents. This is not true, however, for neuropeptide-toxin conjugates, such as dermorphin-SAP. For example, in an unpublished study, we injected large doses (1-2 µg) of dermorphin-SAP into the lumbar intrathecal space of rats. After 2-3 days, rats were sacrificed and lumbar dorsal root ganglia examined for evidence of toxin effect (striking chromatolysis). None was found after examining numerous ganglia and >15,000 primary afferent neurons. Apparently, dermorphin-SAP is not retrogradely transported even if it is taken into the primary afferent terminals that express the mu opioid receptor (MOR).
Q: If a targeted toxin cannot be used in retrograde transport, will it only kill cell bodies in the injection site or will it also kill terminals?
A: Current evidence suggests that applying dermorphin-SAP (Cat. #IT-12) to the population of MOR-expressing neurons in the dorsal horn of the spinal cord results in destruction only of the neurons in lamina II and not the primary afferent terminals that also express MOR. This may be a general principle but it has not been tested in any other situation for dermorphin-SAP, nor have SP-SAP (Cat. #IT-07) and SSP-SAP (Cat. #IT-11) been evaluated for terminal uptake and suicide transport. Any saporin taken into a nerve terminal should not be toxic unless retrogradely transported to the cell body since there are no ribosomes (site of saporin action) or protein synthesis in the nerve terminal.
See: Targeted Toxins
Suggested Reading:
- Oeltmann TN, Wiley RG (1986) Wheat germ agglutinin-ricin A-chain conjugate is neuronotoxic after vagal injection. Brain Res 377:221-228.
- Wiley RG, Stirpe F, Thorpe P, Oeltmann TN (1989) Neuronotoxic effects of monoclonal anti-Thy 1 antibody (OX7) coupled to the ribosome inactivating protein, saporin, as studied by suicide transport experiments in the rat. Brain Res 505:44-54.
- Contestabile A, Fasolo A, Virgili M, Migani P, Villani L, Stirpe F (1990) Anatomical and neurochemical evidence for suicide transport of a toxic lectin, volkensin, injected in the rat dorsal hippocampus. Brain Res 537(1-2):279-286.
- Pangalos MN, Francis PT, Pearson RC, Middlemiss DN, Bowen DM (1991) Destruction of a sub-population of cortical neurones by suicide transport of volkensin, a lectin from Adenia volkensii. J Neurosci Methods 40(1):17-29.
- Wiley RG (1992) Neural lesioning with ribosome-inactivating proteins: suicide transport and immunolesioning. Trends in Neurosci 15:285-290.
- Roberts RC, Harrison MB, Francis SMN, Wiley RG (1993) Differential effects of suicide transport lesions of the striatonigral or striatopallidal pathways on subsets of striatal neurons. Exp Neurol 124:242-252.
- Contestabile A, Stirpe F (1993) Ribosome-inactivating proteins from plants as agents for suicide transport and immunolesioning in the nervous system. Eur J Neurosci 5:1292-1301.
- Wiley RG, Lappi DA(1994) Suicide Transport and Immunolesioning. R.G. Landes, Houston.
- Roberts RC, Strain-Saloum C, Wiley RG (1995) Effects of suicide transport lesions of the striatopallidal or striatonigral pathways on striatal ultrastructure.Brain Res 710:227-237.
- Wiley RG, Kline IVRH (2000) Neuronal lesioning with axonally transported toxins. J Neurosci Methods 103:73-82.
Dermorphin-saporin targets descending facilitation in the rostral ventromedial medulla (rvm) to block CCK-induced abnormal pain
Burgess SE, Gardell LR, Xie Y, Ossipov MH, Vanderah TW, Malan TP, Porreca F, Lai J (2002) Dermorphin-saporin targets descending facilitation in the rostral ventromedial medulla (rvm) to block CCK-induced abnormal pain. Neuroscience 2002 Abstracts 351.11. Society for Neuroscience, Orlando, FL.
Summary: Abnormal pain from L5/L6 spinal nerve ligation (SNL) has been shown to require a time-dependent activation of descending facilitatory pathways arising in the RVM. Additionally, RVM microinjection of L365,260, a cholecystokinin (CCKB) receptor antagonist, reverses SNL-induced tactile and thermal hyperalgesia. These observations suggest the possibility that RVM CCK might “drive” such facilitation from the RVM by activating the endogenous descending facilitation system. Rats were treated with a single RVM injection of dermorphin (DERM) (μ opioid agonist), unconjugated saporin (SAP), or dermorphin-saporin (DERM-SAP) and responses to non-noxious tactile (von Frey filaments) or noxious radiant heat stimuli applied to the hindpaw were measured before and after RVM microinjection of CCK to uninjured rats. RVM DERM-SAP, DERM or SAP did not significantly alter baseline sensory thresholds over 28 days post-injection. At day 28, the rats received bilateral microinjections of CCK (30ng) in the RVM. Rats pretreated with DERM or SAP showed a time-related and revsersible CCK-induced tactile and thermal hypersensitivity. In contrast, RVM CCK failed to produce changes in sensory threshold in animals pretreated with DERM-SAP. The RVM pretreatments did not alter responses in control rats challenged with CCK vehicle. Additionally, lesions of the dorsolateral funiculus also blocked RVM CCK-induced tactile and thermal hypersensitivity. These data support the possibility of CCK-mediated activation of descending facilitation from the RVM as a mechanism of neuropathic pain.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
Enhanced morphine analgeisa after spinal dermorphin-saporin
Miller SA, Lappi DA, Wiley RG (2002) Enhanced morphine analgeisa after spinal dermorphin-saporin. Neuroscience 2002 Abstracts 218.1. Society for Neuroscience, Orlando, FL.
Summary: Dermorphin-saporin (derm-sap) is a neuropeptide toxin conjugate which is selective for neurons expressing the mu-opiate receptor (MOR). The dermorphin moiety of the conjugate binds MOR which is then internalized by the neuron, carrying the toxin with it. The saproin moiety inactivates ribosomes resulting in cell death. In the present study we sought to determine the effect of destroying MOR expressing neurons in Lamina II of the spinal cord dorsal horn on baseline thermal pain sensitivity and response to systemic morphine analgesia. 456 ng derm-sap (n=8) and vehicle (n=8) were injected into the lumbar CSF of adult male Sprague Dawley rats using a subarachnoid catheter inserted through the atlanto-occipital membrane and passed cadually to the level of the lumbar enlargement. 10 minutes following toxin injection, the catheters were withdrawn and the animals allowed to recover. When tested on a hotplate at 52C and on tail-flick assay, toxin rats did not differ from rats injected with vehicle. However, the dose-response curves for subcutaneous morphine were significantly shifted to the left (increased potency) in the toxin treated rats when compared with vehicle controls. Histological analysis of multiple dorsal root ganglia failed to reveal evidence of any primary afferent cell loss. We interpreted these findings to indicate that the neurons destroyed by derm-sap are lamina II MOR expressing neurons and play a role in morphine analgesia at high stimulus intensities.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
Changes in rostral ventromedial medulla (RVM) neurons after the selective loss of mu-opioid receptor expressing cells.
Meng ID, Harasawa I, Lai J, Porreca F, Fields HL (2002) Changes in rostral ventromedial medulla (RVM) neurons after the selective loss of mu-opioid receptor expressing cells. Neuroscience 2002 Abstracts 351.9. Society for Neuroscience, Orlando, FL.
Summary: Different subpopulations of RVM neurons inhibit or facilitate dorsal horn nociceptive transmission. Microinjection of saporin conjugated to the mu-opioid receptor (MOR) agonist dermorphin (derm-sap) into the RVM selectively ablates MOR expressing neurons and diminishes neuropathic pain symptoms (Porreca et al., 2001). We examined the properties of neurons surviving a single RVM injection of derm-sap or sap control. Three classes of RVM neurons (On, Off, and Neutral) have been described with distinct responses to noxious stimuli and MOR agonists. On-cells increase and Off-cells cease firing just prior to a tail flick; MOR agonists inhibit On-cells and disinhibit Off-cells. Neutral cells are unaffected by either noxious stimulation or MOR agonists. Using single unit recording in lightly anesthetized rats a total of 10 electrode tracks were made per rat and each unit encountered was characterized according to its tail flick related activity. Injection of derm-sap (n=8) resulted in fewer On- and Off-cells when compared to saporin controls (n=8). The number of Neutral cells remained unchanged. In separate experiments, after derm-sap pretreatment RVM injections of the MOR agonist DAMGO were ineffective whereas injections of the glutamate receptor agonist homocysteic acid into the same sites increased tail flick latencies. The decrease in number of On-cells after derm-sap is consistent with evidence that these neurons express MOR and facilitate nociceptive transmission. The decrease in number of Off-cells indicates that inhibitory neurons responsible for producing the Off-cell tail flick related pause also express MOR.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
Selective lesion of ventral tegmental area neurons expressing mu-opioid receptors alters EEG power spectrum across sleep/wake cycle
Gallegos RA, Lee RS, Crawford E, Wills DN, Carr JR, Zhukov VI, Slaght KE, Huitron-Resendiz S, Criado JR, Henriksen SJ (2002) Selective lesion of ventral tegmental area neurons expressing mu-opioid receptors alters EEG power spectrum across sleep/wake cycle. Neuroscience 2002 Abstracts 276.14. Society for Neuroscience, Orlando, FL.
Summary: The ventral tegmental area (VTA) has long been implicated in motivated behaviors. Our previous study (Lee et al, J Neurosci 2001) also suggests a role for VTA GABAergic neurotransmission in REM sleep. In the current study the potential role of the VTA in modulating electroencephalogram (EEG) activation was explored by selectively deactivating mu-opioid receptor expressing cells in the VTA. Under sodium pentobarbital anesthesia, rats received either (1) a sham operation (2) a single bilateral VTA injection of NMDA (3) a saporin injection or (4) an injection of a dermorphin-saporin (DERM-SAP) conjugate (Advanced Targeting Systems, San Diego). Animals were also fitted with skull electrodes for recording the EEG. The filtered EEG was recorded continuously for 24 hours beginning 21 to 28 days after surgery. Frequency analysis of the EEG in 15-sec epochs revealed differences in the distribution of relative power in the DERM-SAP animals, compared to controls. Low frequency components (0.5-3.0 Hz and 3.0-8.0 Hz) were enhanced in dual lesioned animals during the dark phase but only during sleep. These results indicate that a selective inactivation of cells in the VTA has specific effects on arousal mechanisms in the intact animal.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
Efferent projections from the striatal patch compartment: anterograde degeneration after selective ablation of neurons expressing mu-opioid receptor in rats.
Tokuno H, Chiken S, Kametani K, Moriizumi T (2002) Efferent projections from the striatal patch compartment: anterograde degeneration after selective ablation of neurons expressing mu-opioid receptor in rats. Neurosci Lett 332(1):5-8. doi: 10.1016/s0304-3940(02)00837-6
Summary: Taking advantage of the fact that neurons in patch compartments of the striatum express µ-opioid receptors, the authors injected 8.5 ng of dermorphin-SAP (Cat. #IT-12) into the striatum of rats. This lesion produced a degeneration of patch neurons as well as anterograde degeneration of efferent fibers from patch compartments, allowing further elucidation of the functional organization of the striatum.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
Descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain.
Burgess SE, Gardell LR, Ossipov MH, Malan T, Vanderah TW, Lai J, Porreca F (2002) Descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain. IASP 2002 Abstracts International Association for the Study of Pain, San Diego, CA.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)