Background Intrathecal morphine forms granulomas that arise from the adjacent arachnoid membrane. We propose that these inflammatory cells exit the meningeal vasculature secondary to meningeal mast cell degranulation. Methods Three sets of experiments were accomplished in dogs. 1) Ex vivo Meningeal mast cell degranulation. Histamine release was measured ex vivo from canine dura incubated with opiates. 2) In vivo cutaneous mast cell degranulation. Flare areas on the dog abdomen were measured after subcutaneous opiates. 3) In vivo granuloma pharmacology. Dogs with lumbar intrathecal catheters received infusion of intrathecal saline or intrathecal morphine. Intrathecal morphine dogs received: i) No other treatment (Control); ii) Twice daily subcutaneous naltrexone; iii) Intrathecal co-infusion of cromolyn; or, iv) Twice daily subcutaneous cromolyn for the 24–28 day study course. Results 1) Morphine but not fentanyl evoked dural histamine release, which was blocked by cromolyn but not naloxone. 2) Wheal/flare was produced by subcutaneous morphine, methadone, hydromorphone, but not fentanyl, and was unaffected by naltrexone but prevented by cromolyn. 3) Granulomas occurred in all dogs receiving intrathecal morphine (15/15); subcutaneous naltrexone had no effect on granulomas (6/6), but was reduced by concurrent intrathecal cromolyn (0/5) or twice daily subcutaneous cromolyn (1 of 5). Conclusions The pharmacology of cutaneous/dural MC degranulation and intrathecal granulomas are comparable, not mediated by opioid receptors, and reduced by agents preventing MC degranulation. If an agent produces cutaneous MC degranulation at concentrations produced by intrathecal delivery, the agent may initiate granulomas.

Intrathecal (IT) gene transfer using adeno-associated virus (AAV) may be clinically promising as a treatment for chronic pain if it can produce sufficiently high levels of a transgene product in the cerebrospinal fluid (CSF). While this strategy was developed in rodents, no studies investigating CSF levels of an analgesic or anti-allodynic protein delivered by IT AAV have been performed in large animals. Interleukin-10 (IL-10) is an anti-allodynic cytokine, for which target therapeutic levels have been established in rats. The present study tested IT AAV8 encoding either human IL-10 (hIL-10) or enhanced green fluorescent protein (EGFP) in a dog model of IT drug delivery. AAV8/hIL-10 at a dose of 3.5×1012 genome copies induced high hIL-10 levels in the CSF, exceeding the target concentration previously found to be anti-allodynic in rodents by >1000-fold. AAV8/EGFP targeted the primary sensory and motor neurons and the meninges. hIL-10, a xenogeneic protein in dogs, induced anti-hIL-10 antibodies detectable in the dogs’ CSF and serum. The high hIL-10 levels demonstrate the efficacy of AAV for delivery of secreted transgenes into the IT space of large animals suggesting a strong case for further development towards clinical testing.

Background We hypothesize that intrathecal (IT) granulomas arising from the IT infusion of several opiates may result from the degranulation of meningeal mast cells (MC). Given functional covariance between cutaneous and meningeal mast cells, we propose that opioids that do not degranulate cutaneous mast cells will not produce a granuloma. An opioid meeting this criteria is the phenylpiperadine Alfentanil HCl. Methods Three experiments were accomplished in dogs. 1) Cutaneous MC degranulation. Flare areas on the dog abdomen were measured after intradermal (ID) alfentanil, morphine or compound 48–80. 2) Dose ranging of analgesic effects of IT alfentanil infusion. Dogs with lumbar IT catheters received continuous infusion for 24 hrs of different concentrations (1–20 mg/mL/d) of alfentanil and analgesic effects were assessed. iii) Granuloma inducing effects. Dogs received IT Alfentanil (20 mg/ml/d; N = 5; 22–28 days)) or morphine (12 mg/mL/d; N=3; 22–30 days) and spinal cord harvested for histopathology after 22–30d of infusion. Results 1) ID Morphine (10 mg/mL) and Compound 48–80 (1mg/mL) but not alfentanil at concentrations up to 20mg/mL produced a cutaneous flare. Intrathecal Alfentanil infusion produced increases in thermal escape latency at concentrations as low as 2 mg/mL/d). A significant depression of arousal was noted in the dogs receiving 20 mg/mL. Over the 22–30 day infusion period, morphine (12 mg/mL/d) resulted in granulomas in all three animals examined whereas intrathecal alfentanil at 20 mg/mL/d failed to initiate a granuloma in any animal. Conclusions These results support the hypothesis linking mast cell degranulation and intrathecal granulomas.

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.