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.
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