Role of p75 Neurotrophin Receptor in Isoflurane-mediated Neuronal Changes

We thank Panni and Panni for their interest in our research article published in Anesthesiology.1The data in that publication provided strong proof that isoflurane neurotoxicity in neonatal rodent pups and in neurons in culture (days in vitro [DIV] 5–7) is mediated at least in part by reduced tissue plasminogen activator release and increased probrain-derived neutrotrophic factor signaling via  the p75 neurotrophic receptors (p75^NTR). This contention is supported by a reduction in tissue plasminogen activator release, increased p75^NTR-mediated c-Jun N-terminal kinase activation, prevention of toxicity by Fc-TrkB (scavenges probrain-derived neutrotrophic factor), and by exogenous tissue plasminogen activator and prevention of toxicity by Pep5 (a specific peptide inhibitor of p75^NTR). Moreover, knockdown of p75^NTRby small interfering ribonucleic acid also mitigated toxicity. Multiple lines of evidence therefore support our contention.

That said, in a comprehensive study, new questions about the possible mechanisms inevitably arise; these serve as impetus for future studies. Panni and Panni have raised several concerns. Apoptosis was evaluated by activated caspase-3 staining only, and other means of identification of apoptotic cells, such as terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining, were not used. The use of activated caspase-3 staining for the detection of apoptosis is well established. Nonetheless, to corroborate the activated caspase-3 data, we also used caspase-activated DNase, a highly specific marker of apoptosis, in our immunoblot studies. In those studies, caspase-activated DNase and activated caspase-3 results were similar. We have also previously used caspase-activated DNase immunofluorescence and the results with this technique are identical to those used from activated caspase-3 staining.2Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining, by contrast, is not specific for apoptosis as deoxyribonucleic acid methylation is observed in cells undergoing necrosis. While additional methods of apoptosis detection would provide some incremental information, the relative value of this information, in so far as the support or refutation of the primary hypothesis is concerned, would be at best limited. We are therefore comfortable with the use of activated caspase-3 and caspase-activated DNase for detection of apoptosis. We also wish to point out that apoptosis was not the only endpoint of the study. Additional immunofluorescence with drebrin staining and electron microscopic analysis revealed the damage at a cytoskeletal/morphologic level. The morphologic alterations induced by anesthesia on developing neurons included p75^NTR-mediated loss in dendritic spines (as indicated by drebrin loss) and morphologic loss of intact synapses, both of which were attenuated by the intracellular p75^NTRinhibitor, TAT-Pep5. When taken in aggregate, our data clearly demonstrate the multiple facets of injury produced by isoflurane.

A second concern is that apoptosis was evaluated within a very narrow window (2 h) after exposure, and injury was not evaluated at later time points. In published studies of anesthetic neurotoxicity, apoptosis is detected early after exposure. In fact, much of apoptosis is not observed 24 h after exposure. The intention of our study was not to repeat the work previously published with respect to the time course of neuronal apoptosis, but to define the underlying molecular mechanisms of injury. With that intent, the selection of a single time point at which a substantial amount of injury is evident is entirely justified.

We agree with Panni and Panni that levels of p75^NTRexpression might account for some of our findings. As indicated by them, p75^NTRexpression decreases with increasing age.3,4The relatively high expression of p75^NTRat postnatal days 5–7 (or DIV 5-7) would make neurons more vulnerable upon anesthetic exposure. The proposed mechanism of p75^NTRexpression changes based on age is interesting from a developmental standpoint, and of course would be strengthened with data revealing the expression profile of p75^NTRin the developing central nervous system. The possibility that isoflurane increases p75^NTRis also of interest, as indicated by Panni and Panni. While the immunoblot data are suggestive of an increase in p75^NTRexpression with isoflurane, we currently do not have definitive data. Unpublished data from our laboratory have indicated that isoflurane neurotoxicity is evident as early as 30 min after exposure in vitro , and this toxicity is abolished by p75^NTRinhibition. This time frame is quite short and argues against the premise that isoflurane increases p75^NTRexpression. Nonetheless, we are in the process of defining not only age-related effects, but also the effect of isoflurane on the expression of p75^NTRin our experimental models.

While total p75^NTRexpression levels are certainly of interest, the precise means by which p75^NTRsignals and its interaction with other partner proteins is just as important. p75^NTR, which is a member of the tumor necrosis factor receptor family, protein expression can increase in pathologic states.5However, p75^NTRcan interact with tropomyosin receptor kinase (Trk) to induce neurite outgrowth and cell survival through either recruitment and transportation of Trk receptors or through enhanced affinity and specificity,6–8or it can induce neuronal apoptosis independent of Trk receptors through alternative signaling pathways.5,9An alternative explanation to age-related reduction in receptor expression is an alteration in the coupling between the p75^NTRand Trk A/B/C receptors, thus moving p75^NTRmore towards prosurvival signaling via  downstream effectors such as Akt, Src or ERK1/2, and further away from a p75^NTR–c-Jun N-terminal kinase-apoptotic pathway. Use of immunoprecipitation experiments at different developmental time points after receptor agonism may explain whether this is an alteration in receptor signaling or changes in receptor expression with age. What does appear to be known is that p75^NTRexpression and signaling is not only temporally but also spatially dependent on some unknown intracellular mechanism. Studies to characterize p75^NTRexpression and its coupling with known partners (e.g. , Trk) at varying ages are currently underway in our laboratory. The expectation is that these studies will provide more detail about the mechanisms by which isoflurane injures developing neurons.

*VA San Diego Healthcare System Anesthesia, San Diego, California. ppatel@ucsd.edu