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Lumbar intrathecal CCK-saporin: anatomic and nociceptive effects
Datta S, Chatterjee K, Kline IV RH, Wiley RG (2008) Lumbar intrathecal CCK-saporin: anatomic and nociceptive effects. Neuroscience 2008 Abstracts 773.4/MM32. Society for Neuroscience, Washington, DC.
Summary: Lumbar intrathecal CCK (cholecystokinin) appears anti-opiate in nocifensive reflex testing and may be important in opiate-resistant neuropathic pain states suggesting a role for CCK receptor-expressing dorsal horn neurons in nociception. In the present study, we sought to determine if selective destruction of CCK receptor-expressing superficial dorsal horn neurons alters pain sensitivity or the analgesic potency of morphine using the targeted cytotoxic conjugate (CCK-sap) of CCK to saporin, a ribosome inactivating protein. 28 adult Sprague Dawley rats were injected via lumbar intrathecal catheter with CCK-sap in doses of 500 ng (n=2), 350 ng (n=3), 700 ng (n=3), 1000 ng (n=4), 1500 ng (n=4), or 3000 ng (n=4). Controls included PBS (n=4) or 1500 ng of plain, unconjugated saporin (n=4). 2 weeks later rats were sacrificed. Lumbar spinal cords were frozen sectioned at 40 µm. One-in-six series of transverse sections at L4-6 were immunostained for CCK. Two rats were injected with 1500 ng of CCK-sap followed by transcardiac aldehyde perfusion in 72 hours. L5 Dorsal root ganglia (DRG) sections were stained with cresyl violet and examined for signs of acute cytotoxicity (chromatolysis and karyohexis). 350 to 1500 ng of intrathecal CCK-sap were well tolerated with no obvious signs of any toxicity. 3000 ng of intrathecal saporin led to motor signs within 72 hours including increased muscle tone, leading to tonic hind limbs extension. Subsequently, twelve Long Evans female rats were tested before and after intrathecal injection of either PBS (n=8) or CCK-sap, 1500 ng (n=4) on: 1 – cold plate (15 °C); 2 – thermal preference shuttle box testing (15/45°C); 3 – hotplate at 44°C, 47°C and 52°C and 4 – thermal preference after morphine (0.5, 1 and 2.5 mg/kg s.c). Anatomical analysis revealed that 1500 ng of CCK-sap decreased CCK immunostaining in the L4-6 Dorsal horn. No acute cytotoxicity was seen in the DRG with1500 ng CCK-sap. Intrathecal CCK-sap was well tolerated at doses ≤1500 ng. CCK-sap produced increased hot side time and decreased crossovers in the thermal preference test. In contrast, CCK-sap decreased latency to first hindpaw lift and increased total responding on the 44 °C hotplate. CCK-sap rats also showed increased hot side time at 45° C at all morphine doses (0, 1 and 2.5 mg/kg s.c.) also with decreased crossovers. We interpret these observations to indicate that CCK-sap produced increased nocifensive reflex responding on the 44° C hotplate consistent with positive modulation of motor responsiveness, and CCK-sap reduced aversion to 45° C heat consistent with an analgesic effect that was additive with morphine.
Related Products: CCK-SAP (Cat. #IT-31)
Neuronal nitric oxide synthase is upregulated in a subset of primary sensory afferents after nerve injury which are necessary for analgesia from alpha2-adrenoceptor stimulation.
Ma W, Eisenach JC (2007) Neuronal nitric oxide synthase is upregulated in a subset of primary sensory afferents after nerve injury which are necessary for analgesia from alpha2-adrenoceptor stimulation. Brain Res 1127(1):52-58. doi: 10.1016/j.brainres.2006.10.008
Summary: Peripheral nerve injury resulting in neuropathic pain often responds poorly to opioid treatment. alpha2-adrenoreceptor (AR) agonists, however, perform better after this type of injury. After a spinal nerve ligation, rats were treated with a 0.6 µg-intrathecal injection of 192-saporin (Cat. #IT-01). The increase of neuronal nitric oxide synthase (nNOS) caused by spinal ligation was abolished in the lesioned animals. The data indicate that AR agonists may reduce sensitization by activating nNOS fibers in the superficial dorsal horn.
Related Products: 192-IgG-SAP (Cat. #IT-01)
Safety evaluation of Intrathecal Substance P-Saporin, a targeted neurotoxin, in dogs.
Allen JW, Mantyh PW, Horais K, Tozier N, Rogers SD, Ghilardi JR, Cizkova D, Grafe MR, Richter P, Lappi DA, Yaksh TL (2006) Safety evaluation of Intrathecal Substance P-Saporin, a targeted neurotoxin, in dogs. Toxicol Sci 91(1):286-298. doi: 10.1093/toxsci/kfj143
Summary: SP-SAP (Cat. #IT-07) has been shown to reverse neuropathic pain behavior in rodents and prevent the formation of hyperalgesia. A safety study was done in beagles to further the use of this molecule as a human therapeutic. Animals received doses from 1.5-150 µg of SP-SAP as bolus intrathecal lumbar injections. Doses of 15 µg and above displayed significant loss of NK1r-expressing cells in lumbar Laminae II and I, but no adverse toxicity was observed at any dose.
Related Products: SP-SAP (Cat. #IT-07)
Pain facilitatory cells in the rostral ventromedial medulla coexpress opioid-μ receptors and cholecystokinin type 2 receptors
Zhang W, Gardell SE, Xie Y, Luo M, Rance NE, Vanderah TW, Porreca F, Lai J (2005) Pain facilitatory cells in the rostral ventromedial medulla coexpress opioid-μ receptors and cholecystokinin type 2 receptors. Neuroscience 2005 Abstracts 394.17. Society for Neuroscience, Washington, DC.
Summary: Pain transmission can be modulated by descending input to the spinal dorsal horn from the rostral ventromedial medulla (RVM). RVM neurons that facilitate nociception are termed “ON-cells”, which are inhibited by mu-opioids, suggesting that they express opioid mu receptors (MOR). Focal application of cholecystokinin (CCK8(s)) into the RVM elicits acute thermal and tactile hypersensitivity and induces ON-cell activity. In situ hybridization using riboprobes for either rat MOR or rat cholecystokinin type-2 receptor (CCK-2) confirms the expression of these receptors in the RVM. Pretreatment with a toxin conjugate, CCK8(s)-saporin results in a significant loss of CCK-2 positive cells in the RVM, concomitant with a blockade of CCK8(s) induced hyperalgesia. The pretreatment also significantly reduces the number of neurons labeled for MOR in the RVM, suggesting that MOR and CCK-2 may be co-localized in some RVM cells. Consistent with these data, similar pretreatment with the toxin conjugate, dermorphin-saporin, which selectively targets MOR expressing neurons, significantly reduces the number of MOR labeled cells in the RVM, blocks RVM CCK8(s) induced hyperalgesia and reduces the number of CCK-2 positive cells in the RVM. In situ hybridization using 35S-labeled CCK-2 riboprobes and Digoxigenin-labeled MOR riboprobes shows that over 80% of labeled RVM neurons co-express both MOR and CCK-2, ~15% express only CCK-2, and very few cells express only MOR. These findings represent the first direct demonstration of the phenotype of pain facilitatory neurons in the RVM. Together with previous studies showing that RVM CCK-2 antagonists reverse nerve injury-induced pain, this phenotype provides strong support for the view that endogenous CCK is a critical mediator of the descending pain facilitation, particularly in the maintenance of experimental neuropathic pain. Support Contributed By: NIDCR R01 DE016458
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), CCK-SAP (Cat. #IT-31)
Spinal-supraspinal serotonergic circuits regulating neuropathic pain and its treatment with gabapentin.
Suzuki R, Rahman W, Rygh LJ, Webber M, Hunt SP, Dickenson AH (2005) Spinal-supraspinal serotonergic circuits regulating neuropathic pain and its treatment with gabapentin. Pain 117(3):292-303. doi: 10.1016/j.pain.2005.06.015
Summary: The anticonvulsant, gabapentin, is thought to modulate calcium channel function. In animals, it also affects abnormal pain function. 10 µl of 1 µM SP-SAP (Cat. #IT-07) was injected into the subarachnoid space of rats. It was found that the effects of gabapentin were blocked when NK-1r expressing neurons in the dorsal horn were eliminated. The results suggest that not only is the NK-1r pathway a determinant of neuronal and behavioral manifestations of neuropathy, it is also involved in the action of gabapentin.
Related Products: SP-SAP (Cat. #IT-07)
The behavioral and neuroanatomical effects of IB(4)-saporin treatment in rat models of nociceptive and neuropathic pain.
Tarpley JW, Kohler MG, Martin WJ (2004) The behavioral and neuroanatomical effects of IB(4)-saporin treatment in rat models of nociceptive and neuropathic pain. Brain Res 1029(1):65-76. doi: 10.1016/j.brainres.2004.09.027
Summary: Using the fact that primary afferent neurons bind isolectin B4 (IB4), the authors injected 5 µg of IB4-SAP (Cat. #IT-10) into the sciatic nerve in the left thigh. After recovery, these animals were then treated with a L5 spinal nerve ligation. Lesioned animals displayed attenuated NGF-induced hyperalgesia, as well as differences in other pain-model markers. The data indicate that IB4-positive C-fibers play a discrete role in NGF-induced hyperalgesia, as well as in the development of neuropathic pain.
Related Products: IB4-SAP (Cat. #IT-10)
Selective elimination of mu-opioid receptor expressing neurons in the rostral ventromedial medulla (RVM) does not affect periaqueductal gray (pag) stimulation-produced analgesia
Harasawa I, Lai J, Porreca F, Fields HL, Meng ID (2003) Selective elimination of mu-opioid receptor expressing neurons in the rostral ventromedial medulla (RVM) does not affect periaqueductal gray (pag) stimulation-produced analgesia. Neuroscience 2003 Abstracts 177.5. Society for Neuroscience, New Orleans, LA.
Summary: PAG stimulation produces antinociception at spinal levels by modulating RVM neuronal activity. Microinjection of saporin conjugated with the mu-opioid receptor agonist dermorphin (DERM-SAP) into the RVM selectively eliminates MOR expressing neurons and diminishes neuropathic pain symptoms (Porreca et al., 2001). The aim of the present study was to determine whether MOR expressing neurons in the RVM are required for PAG stimulation produced analgesia (PAG/SPA). The minimum electrical current required to inhibit the tail flick response was compared in barbiturate-anesthetized rats given a single RVM injection of SAP or DERM-SAP 3-4 weeks prior to testing. Thresholds in SAP and DERM-SAP treated rats were not different. Furthermore, microinjection of the glutamate receptor antagonist kynurenic acid (10 mM, 800 nl) into the RVM disrupted PAG/SPA in both SAP and DERM-SAP treated rats. These results indicate that 1) mu-receptor expressing neurons in the RVM are not necessary for PAG/SPA, and 2) excitatory amino acid transmission in the RVM is critical for PAG/SPA. In additional experiments, inhibition of neurotransmitter release in the RVM by the microinjection of cobalt chloride (CoCl2, 100 mM, 800 nl), produced significant antinociception only in DERM-SAP treated rats. This finding suggests that DERM-SAP injections result in increased tonic inhibition of RVM neurons and that CoCl2 disinhibits these neurons to produce antinociception. Tonic inhibition of off-cells would account for our failure to find off-cells in DERM-SAP treated rats.
Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)
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)
Long-term effects on pain behavior of decreased spinal noradrenaline in neuropathic rats
Ohara PT, Boudah A, Jasmin L (2002) Long-term effects on pain behavior of decreased spinal noradrenaline in neuropathic rats. Neuroscience 2002 Abstracts 351.22. Society for Neuroscience, Orlando, FL.
Summary: We sought to determine if a permanent reduction in the noradrenergic (NA) input to the spinal cord in adult rats would alter the pain behavior associated with nerve injury. Selective NA denervation of the lumbo-sacral cord was achieved by intrathecal injection of anti-dopamine beta-hydroxylase antibodies conjugated to the toxin saporin in 12 female rats. Spinal NA denervation was confirmed histologically in all animals. Saline injected rats served as controls. Two weeks after toxin or saline treatment, a unilateral peripheral neuropathy was induced by tight ligation of the left L5 spinal nerve in both groups. Unexpectedly, the same degree of mechanical hyperalgesia was present in the neuropathic paw of rats in both the toxin and saline treated groups. Rats lacking NA spinal afferents, however, were less responsive to the antinoiceptive effects of morphine administered systemically or intracerebroventricularly. Also, toxin treated rats did not display opioid dependant stress analgesia. Finally, toxin treated rats were more responsive to the antinociceptive effect of the NK1 antagonist CP 96,345 but not to its enantiomer CP 96,344. From these results we conclude that the permanent loss of spinal NA does not alter neuropathic pain behavior, possibly because of compensatory changes in the CNS. The decreased response to opioids is consistent with the previous suggestions of an interaction between noradrenergic and opioidergic systems in producing analgesia. The increased response to NK1 antagonists shows that NA tonically inhibits substance.
Related Products: Anti-DBH-SAP (Cat. #IT-03)
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)