No abstract

Subarachnoidal grafting of monoamine-producing cells has been used with success to treat chronic pain in animal models. In the search for a source of autologous transplantable cells, capable of delivering neuroactive substances to the cerebrospinal fluid (CSF) to treat pain, we have tested adipose tissue-derived stromal cells (ADSCs) transduced to produce levodopa. Intrathecally grafted ADSCs survive for long term adhered to spinal cord and nerve root meninges. Cultured ADSCs were retrovirally transduced with tyrosine hydroxylase (TH) and/or GTP cyclohydroxylase 1 (GCH1) genes and stably expressed them for at least 6 weeks in culture. Singly transduced cultures did not produce measurable levodopa but doubly transduced or a mixture of singly transduced ADSCs were able to efficiently synthesize and release levodopa. When 0.5-1 × 10 6 THand GCH1-expressing ADSCs were intrathecally grafted in rats, elevated levels of levodopa and dopamine metabolites were found in CSF at 3 days, although at lower concentrations than expected. Unexpectedly, no levodopa was measurable in CSF at 6 days. In a rat model of neuropathic pain, intrathecal grafting of doubly transduced cells did not produce antiallodynic effects at 2 or 6 days, even when histological analysis revealed the presence of weak TH-immunoreactive subarachnoidal cell clusters. These results suggested that doubly transduced cells could indeed function as biological minipumps to enhance the dopaminergic neurotransmission at the spinal cord level but transgenes were rapidly silenced after intrathecal grafting. Transgene silencing was mimicked in culture by serum deprivation for 3 days. Serum addition at this point recovered transgene expression in just 6 h, as did, to a smaller degree, dbcAMP or histone deacetylase inhibitors. Transgene expression silencing in serum deprivation conditions was prevented by 5′-terminal IRES sequences. The present study does not discard the use of transduced cells as a strategy to treat chronic pain but shows that controlling transgene silencing in implanted cells needs to be achieved first.Key words: Levodopa; Neuropathic pain; Intrathecal grafting; Analgesia; Transgene silencing; Rats INTRODUCTIONrenal medulla induced analgesia in rats. Thereafter, chromaffin cells have been extensively tested in animal models of neuropathic pain (8,21,49) obtaining pain reNeuropathies of varied etiology often cause chronic pain that can be difficult to treat. Allodynias (pain triglief. Nevertheless, it is not known yet if the analgesic properties of chromaffin cells can be attributed to their gered by innocuous stimuli), dysesthesias (abnormal, unpleasant sensations), or hyperalgesia (increased sensisecretion of monoamines, opioids, GABA, or even neurotrophic factors (13). Analgesia has been reportedly obtivity to pain) are common symptoms of painful neuropathies, producing great discomfort in patients. Antidetained also by intrathecal implants of opioid-releasing cells (46,47), serotonergic cells (15), and GABAergic pressants, anticonvulsants, opioids...