Intrathecal substance P-saporin in the dog: distribution, safety, and spinal neurokinin-1 receptor ablation.

BACKGROUND: Neurokinin-1 receptors (NK1-rs) located on superficial dorsal horn neurons are essential for integration of nociceptive input. Intrathecal injection of substance P-saporin (SP-SAP) leads to local loss of spinal NK1-r (+) neurons suggesting its potential as a therapeutic agent for chronic pain. The authors determined, in a canine model, effects of lumbar intrathecal SP-SAP. METHODS: Distribution of SP-SAP and Saporin was determined in plasma, lumbar cerebrospinal fluid, and tissue. Safety of intrathecal SP-SAP was determined in four groups (six dogs each) administered 0 (0.9% saline), 1.5, 15, or 150 µg SP-SAP through lumbar intrathecal catheters. Behavioral, physiologic, and biochemical variables were assessed. Spinal tissues were collected at 7 and approximately 90 days, or earlier if significant morbidity developed, and analyzed for NK1-r (+) neuron loss and histopathology. RESULTS: SP-SAP and Saporin were detectable in lumbar cerebrospinal fluid for up to 4 and 24 h, respectively. Animals receiving intrathecal saline, 1.5, or 15 µg of SP-SAP showed no persistent neurologic deficits. Three animals receiving 150 µg of SP-SAP developed pelvic limb paraparesis and were euthanized prematurely. Immunohistochemistry and in situ hybridization cell counts confirmed a significant reduction in NK1-r (+) in superficial dorsal horn neurons from lumbar spinal cord after intrathecal administration of 15 and 150 µg of SP-SAP. A significant loss of NK1-r neurons in the lumbar ventral horn occurred only with 150-µg SP-SAP. CONCLUSION: Intrathecal 15-µg SP-SAP reduced dorsal, but not ventral, NK1-r (+) neurons at the spinal level of delivery with minimal side effects, whereas 150-µg SP-SAP resulted in motor neuron toxicity.

Streptavidin-Saporin: Converting Biotinylated Materials into Targeted Toxins.

Streptavidin-Saporin can be considered a type of 'secondary' targeted toxin. The scientific community has taken advantage of this conjugate in clever and fruitful ways using many kinds of biotinylated targeting agents to send saporin into a cell selected for elimination. Saporin is a ribosome-inactivating protein that causes inhibition of protein synthesis and cell death when delivered inside a cell. Streptavidin-Saporin, mixed with biotinylated molecules to cell surface markers, results in powerful conjugates that are used both in vitro and in vivo for behavior and disease research. Streptavidin-Saporin harnesses the 'Molecular Surgery' capability of saporin, creating a modular arsenal of targeted toxins used in applications ranging from the screening of potential therapeutics to behavioral studies and animal models. The reagent has become a well-published and validated resource in academia and industry. The ease of use and diverse functionality of Streptavidin-Saporin continues to have a significant impact on the life science industry.

Saporin as a Commercial Reagent: Its Uses and Unexpected Impacts in the Biological Sciences-Tools from the Plant Kingdom.

Saporin is a ribosome-inactivating protein that can cause inhibition of protein synthesis and causes cell death when delivered inside a cell. Development of commercial Saporin results in a technology termed 'molecular surgery', with Saporin as the scalpel. Its low toxicity (it has no efficient method of cell entry) and sturdy structure make Saporin a safe and simple molecule for many purposes. The most popular applications use experimental molecules that deliver Saporin via an add-on targeting molecule. These add-ons come in several forms: peptides, protein ligands, antibodies, even DNA fragments that mimic cell-binding ligands. Cells that do not express the targeted cell surface marker will not be affected. This review will highlight some newer efforts and discuss significant and unexpected impacts on science that molecular surgery has yielded over the last almost four decades. There are remarkable changes in fields such as the Neurosciences with models for Alzheimer's Disease and epilepsy, and game-changing effects in the study of pain and itch. Many other uses are also discussed to record the wide-reaching impact of Saporin in research and drug development.

Safety evaluation of intrathecal substance P-saporin, a targeted neurotoxin, in dogs.

Intrathecal (IT) substance P-Saporin (SP-SAP), a 33-kDa-targeted neurotoxin, produces selective destruction of superficial neurokinin 1 receptor (NK1r)-bearing cells in the spinal dorsal horn. In rats, SP-SAP prevents the formation of hyperalgesia and can reverse established neuropathic pain behavior in rodents. To determine the safety of this therapeutic modality in a large animal model, beagles received bolus IT lumbar injections of vehicle, SP-SAP (1.5, 15, 45, or 150 microg), or a nontargeted preparation of saporin (SAP, 150 microg) for immunohistological analysis of spinal cords. Doses of 15 microg SP-SAP and above produced a significant and equivalent loss of NK1r-bearing cells and dendrites in lumbar laminae II and I compared to vehicle- or SAP-treated animals. Cervical regions in all animals displayed no loss of NK1r immunoreactivity as compared to controls. Total numbers of neurons in the lumbar dorsal horn or alpha-motor neurons in the ventral horn demonstrated no significant changes. No increases in the astrocytic marker glial fibrillary acidic protein were noted following treatment with SP-SAP, suggesting a lack of generalized neurotoxicity. Additional dogs received doses of 1.5-150 microg SP-SAP or SAP and were sacrificed after 28 or 90 days to assess behavioral and physiological parameters. Although some acute motor signs were observed with both SP-SAP and SAP, no long-lasting significant events were noted in any of these animals. These data indicate no adverse toxicity at doses up to 10 times those necessary for producing loss of superficial NK1r-bearing neurons in a large animal model.

Targeted toxins in pain.

Although only recently applied to the study of nociception, 'molecular neurosurgery', producing highly selective neural lesions using targeted cytotoxins, has proven a valuable tool for analysis of nociceptive systems and promises to yield much more information on the role of specific types of neurons in pain perception and possibly new pain therapies. Neuropeptide-toxin conjugates, particularly, substance P-saporin, have proven useful research tools and may find clinical applications. Targeting non-lethal moieties (enzymes, genes, viruses) also may prove useful for research and therapeutic purposes.

Medullary serotonergic neurones and adjacent neurones that express neurokinin-1 receptors are both involved in chemoreception in vivo.

Neurokinin-1 receptor (NK1R)-expressing neurones that are involved in chemoreception at the retrotrapezoid nucleus (Nattie & Li, 2002b) are also prominent at locations that contain medullary serotonergic neurones, which are chemosensitive in vitro. In medullary regions containing both types, we evaluated their role in central chemoreception by specific cell killing. We injected (2 x 100 nl) (a) substance P-saporin (SP-SAP; 1 microm) to kill NK1R-expressing neurones, (b) a novel conjugate of a monoclonal antibody to the serotonin transporter (SERT) and saporin (anti-SERT-SAP; 1 microm) to kill serotonergic neurones, or (c) SP-SAP and anti-SERT-SAP together to kill both types. Controls received IgG-SAP injections (1 microm). There was no double-labelling of NK1R-immunoreactive (ir) and tryptophan-hydroxylase (TPOH)-ir neurones. Cell (somatic profile) counts showed that NK1R-ir neurones in the SP-SAP group were reduced by 31%; TPOH-ir neurones in the anti-SERT-SAP group by 28%; and NK1R-ir and TPOH-ir neurones, respectively, in the combined lesion group by 55% and 31% (P < 0.001; two-way ANOVA; P < 0.05, Tukey's post hoc test). The treatments had no significant effect on sleep/wake time, body temperature, or oxygen consumption but all three reduced the ventilatory response to 7% inspired CO(2) in wakefulness and sleep by a similar amount. SP-SAP treatment decreased the averaged CO(2) responses (3, 7 and 14 days after lesions) in wakefulness and sleep by 21% and 16%, anti-SERT-SAP decreased the responses by 15% and 18%, and the combined treatment decreased the responses by 12% and 12% (P < 0.001; two-way ANOVA; P < 0.05, Tukey's post hoc test). We conclude that separate populations of serotonergic and adjacent NK1R-expressing neurones in the medulla are both involved in central chemoreception in vivo.

Spinal neurons involved in the generation of at-level pain following spinal injury in the rat.

Using a conjugate of substance P and the ribosome-inactivating protein saporin, neurons expressing the neurokinin-1 receptor in lamina I of the spinal cord were targeted to determine their role in the expression of a spontaneous pain behavior following intraspinal injections of quisqualic acid in the rat. Treatment was carried out at the time of injury in order to prevent the onset of the behavior, and following onset in order to evaluate the potential clinical utility of this intervention. Treatment at the time of injury resulted in significant decreases in onset-time and severity of pain behavior, while treatment at the time of onset led to a significant reduction of the spontaneous self-directed behavior. The results suggest that the substrate for at-level pain following spinal cord injury includes a population of spinal neurons expressing the neurokinin-1 receptor in the superficial laminae of the spinal cord.

Cholinergic depletion in the nucleus accumbens: effects on amphetamine response and sensorimotor gating.

A delicate balance between dopaminergic and cholinergic activity in the ventral striatum or nucleus accumbens (N.Acc) appears to be important for optimal performance of a wide range of behaviours. While functional interactions between these systems are complex, some data suggest that acetylcholine in the N.Acc. may dampen the effects of excessive dopamine (DA) release. We proposed that a reduction in the density of cholinergic interneurons in the N.Acc would result in behavioural alterations suggestive of a hyper-responsiveness of the N.Acc DA system. The present study aimed to produce a sustainable depletion of cholinergic neurons in the N.Acc in the rat and study the effects of such lesions on DA-dependent behaviour. A novel saporin immunotoxin targeting choline acetyltransferase was microinjected bilaterally into the N.Acc of adult rats. We confirmed histologically that two weeks post-injection, animals show a local, selective depletion of cholinergic interneurons (mean cell loss of 44%). Cholinergic-depleted rats showed a marked increase in the locomotor activating effects of amphetamine. In addition, such lesions induced a disruption of sensorimotor gating processes, reflected in a reduction in the prepulse inhibition of the acoustic startle response, which was reversed by haloperidol. These data are suggestive of pronounced hyper-responsiveness of the meso-accumbens DA system which may be of relevance to the pathophysiology of schizophrenia, a condition where selective reduction in the number of ventral striatal cholinergic neurons has been demonstrated.

Targeted destruction of photosensitive retinal ganglion cells with a saporin conjugate alters the effects of light on mouse circadian rhythms.

Non-image related responses to light, such as the synchronization of circadian rhythms to the day/night cycle, are mediated by classical rod/cone photoreceptors and by a small subset of retinal ganglion cells that are intrinsically photosensitive, expressing the photopigment, melanopsin. This raises the possibility that the melanopsin cells may be serving as a conduit for photic information detected by the rods and/or cones. To test this idea, we developed a specific immunotoxin consisting of an anti-melanopsin antibody conjugated to the ribosome-inactivating protein, saporin. Intravitreal injection of this immunotoxin results in targeted destruction of melanopsin cells. We find that the specific loss of these cells in the adult mouse retina alters the effects of light on circadian rhythms. In particular, the photosensitivity of the circadian system is significantly attenuated. A subset of animals becomes non-responsive to the light/dark cycle, a characteristic previously observed in mice lacking rods, cones, and functional melanopsin cells. Mice lacking melanopsin cells are also unable to show light induced negative masking, a phenomenon known to be mediated by such cells, but both visual cliff and light/dark preference responses are normal. These data suggest that cells containing melanopsin do indeed function as a conduit for rod and/or cone information for certain non-image forming visual responses. Furthermore, we have developed a technique to specifically ablate melanopsin cells in the fully developed adult retina. This approach can be applied to any species subject to the existence of appropriate anti-melanopsin antibodies.