Today’s topic is time-course for our saporin conjugates. Common questions we get are (1) how long does it takes to see cell death, (2) how long should I wait before performing histology on an animal, or (3) how long before I see behavioral changes in an animal?
In this image from Mantyh et al. (1997), we are looking at confocal imagery of the binding and internalization of our peptide conjugate SP-SAP to the NK1r receptor in primary cultures of neonatal spinal cord neurons.
As you can see, conjugate binding occurs immediately where within hours, the SP-SAP has recognized and bound to the NK1 receptor. Here we see the areas of concentrated NK1R expression marked by yellow immunofluorescence.
But how long until you see cell death? Here is a cytotoxicity graph of in vitro data of our antibody conjugate 192-IgG-SAP. These are typical data after cells have been treated for 3 days, which is standard protocol.
Waite et al. (1994) used 192-IgG-SAP and showed the appearance of behavioral effects associated with neuronal loss at day four and plateauing at day 7.
This coincides with the time course seen in vitro. At this point, microglia will infiltrate, but stop at 7 days, which is probably the peak day for infiltration. Once there is complete removal of the detritus, microglia down-regulate and at 14 days all debris is cleared and the animal begins to regain normal eating and sleeping habits. This is the idea behind waiting about 2 weeks before performing histology.
Janes TA, Parra Sanchez AS, Cardani S, Pagliardini S (2025) Progestin infusion into the hypothalamus of CO2-chemoreflex impaired female rats enhances the hypercapnic ventilatory response. Am Physiol Soc 40(S1) doi: 10.1152/physiol.2025.40.S1.1430
Objective: Authors hypothesized that etonogestrel (ETO) infusion into the dorsomedial hypothalamus (DMH) would elicit CO2-chemo reflex recovery similar to that observed in systemically treated female rats.
Summary: The hypothesis was tested by impairing the CO2-chemoreflex in adult female rats by lesioning retrotrapezoid nucleus (RTN) neurons with substance P-conjugated saporin toxin. Preliminary data suggest that ETO induced modest CO2-chemoreflex recovery in rats with moderate-sized RTN lesion by increasing tidal volume and breathing frequency, while hypoxic ventilation was unaffected. In rats with large RTN lesion, no restorative effect was observed. These data suggest a potential ETO-induced compensatory role for DMH neurons following central CO2-chemoreflex impairment but do not rule out contributions from other brainstem nuclei based on previous systemic ETO administration.
Usage: The CO2-chemoreflex was impaired in adult female Sprague-Dawley rats by lesioning RTN neurons with substance P-saporin toxin (SP-SAP).
Objective: To uncover the role of a sparse but unusual population of genetically-distinct interneurons known as type-I nNOS neurons, using a novel pharmacological strategic to unilaterally ablate these neurons from the somatosensory cortex of mice.
Summary: Region-specific ablation produced changes in both neural activity and vascular dynamics, decreased power in the delta-band of the local field potential, reduced sustained vascular responses to prolonged sensory stimulation, and abolished the post-stimulus undershoot in cerebral blood volume. Coherence between the left and right somatosensory cortex gamma-band power envelope and blood volume at ultra-low frequencies was decreased, suggesting type-1 nNOS neurons integrate long-range coordination of brain signals. Authors also observed decreases in the amplitude of resting-state blood volume oscillations and decreased vasomotion following the ablation of type-I nNOS neurons.
Usage: Type-I nNOS neurons were selectively ablated with saporin conjugated to a substance P analog (SP-SAP, IT-07). Intracortical injection with 4 ng of either SAP conjugate or Blank-SAP (IT-21).
Kudsi SQ, Viero FT, Pereira LG, Trevisan G (2024) Involvement of the transient receptor channels in preclinical models of musculoskeletal pain. Curr Neuropharmacol 22(1):72-87. doi: 10.2174/1570159X21666230908094159 PMID: 37694792
Objective: To provide an updated view of the most studied preclinical models of muscle hyperalgesia and the role of transient receptor potential (TRP) in these models.
Summary: The participation of TRPV1, TRPA1, and TRPV4 in different models of musculoskeletal pain was evaluated using pharmacological and genetic tools. All the studies detected the antinociceptive effect of respective antagonists or reduced nociception in knockout mice. Hence, TRPV1, TRPV4, and TRPA1 blockers could potentially be utilized in the future for inducing analgesia in muscle hypersensitivity pathologies.
Usage: NK1-expressing neurons were ablated by SP-SAP (neurotoxin saporin conjugated to substance P) to Vc, and the ablation of NK1-expressing second-order neurons reduced the MGS and bite force nociceptive behaviors.
Díaz-Jara E, Pereyra K, Vicencio S, Olesen MA, Schwarz KG, Toledo C, Díaz HS, Quintanilla RA, Del Rio R (2024) Superoxide dismutase 2 deficiency is associated with enhanced central chemoreception in mice: Implications for breathing regulation. Redix Biol 69:102992. doi: 10.1016/j.redox.2023.102992 PMID: 38142585
Objective: To determine the impact of partial deletion of SOD2 expression on (1) O2.−accumulation in the Retrotrapezoid nucleus (RTN), (2) central ventilatory chemoreflex function, and (3) disordered-breathing as well as cellular localization of SOD2 in the RTN of healthy mice.
Summary: Results showed that SOD2+/− mice displayed reductions in SOD2 levels and high O2-formation and mitochondrial dysfunction within the RTN compared to wild type. Additionally, SOD2+/−mice displayed a heightened ventilatory response to hypercapnia and exhibited overt signs of altered breathing patterns.
Usage: Recent research has shown that removing some RTN chemoreceptor neurons using substance P-conjugated saporin toxin can normalize central chemoreflex and respiratory disorders in animals with heart disease, highlighting the significance of RTN neurons in the pathological process of central chemoreflex potentiation.
Janes TA, Cardani S, Saini JK, Pagliardini S (2024) Etonogestrel promotes respiratory recovery in an in vivo rat model of central chemoreflex impairment. Acta Physiol (Oxf) e14093. doi: 10.1111/apha.14093 PMID: 38258900
Objective: Examine the use of progestins and synthetic progestins in the stimulation of breathing, especially after chemoreflexive impairment.
Summary: Central CO2 chemoreflex is important for respiratory control. The retrotrapezoid nucleus is involved in CO2 chemosensitivity where its removal or inhibition attenuates CO2 chemoreflexes and diminishes restful breathing. Progesterone stimulates restful breathing and CO2 chemoreflexes. The authors investigated whether acute or chronic administration of the progestinic drug, etonogestrel, could help in the recovery of respiratory chemoreflexes following lesion of the retrotrapezoid nucleus via a SP-SAP.
Usage: Rats were injected with 26-43.3 ng/ul of SP-SAP (IT-11) or 46.7 ng/ul of Blank-SAP (IT-21), with 150 nl per injection.
Maiarù M, Leese C, Silva-Hucha S, Fontana-Giusti S, Tait L, Tamagnini F, Davletov B, Hunt SP (2024) Substance P-botulinum mediates long-term silencing of pain pathways that can be re-instated with a second injection of the construct in mice. J Pain 25(6):104466. doi: 10.1016/j.jpain.2024.01.331 PMID: 38218509
Summary: The authors discuss how Substance P-Botulinum is used to try to replicate the permanent results achieved with Substance P-Saporin (SP-SAP, SSP-SAP).
Yaksh TL, dos Santo G, Lemes J, Malange K (2023) Neuraxial drug delivery in pain management: An overview of past, present, and future. Anaesthesiology 37(2):243-265. doi: 10.1016/j.bpa.2023.04.003 PMID: 37321769
Objective: Review of neuraxial therapeutic delivery platforms for pain management and the regulation caused by pharmacological targeting of dorsal root ganglion and dorsal horn systems.
Summary: Studies have shown modulation by spinal opiates, but subsequent work reveals the complexity of the neuraxial system. Various delivery platforms, such as viral transfection, antisense and targeted neurotoxins give evidence to approaches to selectively address the acute and chronic pain phenotype. Substance P-Saporin is discussed as a method to lesion NK1R-containing neurons for the treatment of chronic pain.
Puente BR (2021) Chronic pain in dogs (Dolor crónico en el perro). Zaragoza Spain: Gruppo Asis Biomedia, S. L..
Summary: The author presents a thorough overview of aspects of canine chronic pain. He includes SP-SAP (Substance P-Saporin) as an experimental drug, “its use as an adjuvant analgesic in dogs with bone cancer has been studied,”
Vetter I (2018) Toxins in Neurobiology: New tools from old molecules. Neurosci Lett 679:1-3. doi: 10.1016/j.neulet.2018.05.008
Summary: The selective ablation of neurokinin-1 receptor-expressing neurons by SP-SAP revealed a key role for the preBötzinger complex in the generation of respiratory rhythm. Toxin-mediated neuronal ablation may also find therapeutic applications, such as the treatment of severe refractory pain in terminally ill patients by intrathecal SP-SAP which causes selective loss of neurokinin-1 receptor-expressing neurons in the spinal cord dorsal horn.
