Changes in GAD- and GABA- immunoreactivity in the spinal dorsal horn afterperipheral nerve injury and promotion of recovery by lumbar transplant of immortalized serotonergic precursors

We have utilized RN46A cells, an immortalized neuronal cell line derived fr om E13 brainstem raphe, as a model for transplant of bioengineered serotone rgic cells. RN46A cells require brain-derived neurotrophic factor (BDNF) fo r increased survival and serotonin (5HT) synthesis in vitro and in vivo. RN 46A cells were transfected with the rat BDNF gene, and the 46A-B14 cell lin e was subcloned. These cells survive longer than 7 weeks after transplantat ion into the subarachnoid space of the lumbar spinal cord and synthesize 5H T and BDNF. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the affected hindpaw in rats. Transp lants of 46A-B14 cells placed 1 week after CCI alleviated chronic neuropath ic pain, while transplants of 46A-V1 control cells, negative for 5HT and wi thout the BDNF gene, had no effect on the induction of thermal and tactile nociception. When endogenous cells of the dorsal horn which contain the neu rotransmitter gamma-aminobutyric acid (GABA) and its synthetic enzyme gluta mate decarboxylase (GAD) were immunohistochemically quantified in the lumba r spinal cord 3 days and 1-8 weeks after CCI, the number of GABA- and CAD-i mmunoreactive (ir) cells decreased bilateral to the nerve injury as soon as 3 days after CCI. At 1 week after CCI, the number of GABA-ir cells continu ed to significantly decline bilaterally, returning to near normal numbers o n the side contralateral to the nerve injury by 8 weeks after the nerve inj ury. The number of GAD-ir cells began to increase bilaterally to the nerve injury at I week after CCI and continued to significantly increase in numbe rs over normal values by 8 weeks after the nerve injury. When examined 2 an d 8 weeks after CCI plus cell transplants, the transplants of 46A-B14 cells reversed the increase in GAD-ir cell numbers and the decrease in GABA-ir c ells by 1 week after transplantation, while 46A-V1 control cell transplants after CCI had no effect on the changes in numbers of GAD-ir or GABA-ir cel ls. Collectively, these data suggest that altered 5HT levels, and perhaps B DNF secretion, related to the transplants ameliorate chronic pain and rever se the induction and maintenance of an endogenous pain mechanism in the dor sal horn. This induction mechanism is likely dependent on altered GAD regul ation and GABA synthesis, initiated by CCI. (C) 1998 Elsevier Science B.V. an rights reserved.