The present study was designed to assess whether exposure to repetitive injections of fentanyl during brain development influences later physical and neurological outcomes. Using a neonatal postoperative pain model, this study demonstrates, for the first time, lasting effects on growth and behavior of rat pups that underwent repeated fentanyl exposure during early postnatal life, when tested as later pre-weanling rats. The results showed that repeated fentanyl exposure of an immature stressed animal significantly interferes with growth, cognitive function, behavioral reactivity to stress, neuromuscular and locomotor development, and balance and coordination. All these outcomes () suggest a neurological impact with possible consequences, either positive or negative, later in life.
To examine the role of fentanyl administration in the development of behaviors that occur following repeated exposure to this medication, both in immature CNS and pain settings, we combined two strategies: the model for the study of neonatal neurodevelopment (6-day-old rat pup) was combined with the postincisional pain of Brennan paw incision [14
], as a model for neonatal neurodevelopmental and postoperative pain. Translation of developmental ages from rodents to humans continues to be debated. A review paper by Vidair [19
], which discusses the adequacy of the postnatal rat to serve as a model for neurodevelopment in the postnatal human, concludes that the rat in the third postnatal week is the neurodevelopmental equivalent of the newborn human and that the two species share numerous pathways of postnatal neurodevelopment. Therefore, our neonatal rat model roughly corresponds to a human premature. Brennan's model of incisional pain [14
] was chosen since it simulates the usual clinical setting involving critically ill prematures in neonatal intensive care units.
Premature newborns typically present a broad range of comorbidities which make them a complex group to study, given the many variables, painful/stressful procedures, and pharmacologic exposures involved. Therefore, experimental studies using animals allow us to exclude potential confounding variables. In our study we used a model without comorbidities in a postoperative pain and stress setting. Such a preclinical model, which leads to pain-related events that mirror the symptoms observed in patients undergoing surgery [20
], gives us the opportunity to explore whether repetitive fentanyl exposure, early in neonates subject to painful stimuli, leads to later neurodevelopmental anomalies. The postoperative pain model we used was previously described by Brennan and coworkers [14
]. This rat model consists of an incision of the plantar paw skin, with damage of the underlying muscle, which results in localized mechanical hypersensitivity that lasts 3–5 days. Further research by Brennan's group showed release of excitatory amino acids, such as glutamate and aspartate, activation of dorsal horn cells, and central sensitization [21
Concerning protocol design, the dose of fentanyl used in this study, although at first sight much higher than the neonatal human recommended dose, was chosen according to the species known metabolism to relate to that typically encountered in clinical settings reflecting antinociceptive ED50 values for PND6 rats [22
]. We assessed behavioral problems in our neonatal stressed model using a validated set of tests usually chosen for drug toxicity screening.
Among major findings in the present study, we highlight the significant enhancement of weight gain in fentanyl group compared to controls, as summarized in . Neither fentanyl nor control conditions had significant effects on normal early pup weight gain. In contrast, there were significant group differences in rat weights on PND 21. Rats in the fentanyl group weighed more than those in the saline and unoperated sham groups, with the difference becoming significant around PND 12 and expanding as the pups aged until weaning. These outcomes suggest that the effects of the early postnatal exposure were subtle but, nonetheless, predisposed the pups to abnormal weight gain. Many hypotheses are possible to explain this finding, namely, metabolic derangements, behavior anomalies related to eating disorders, or decreased physical activity. An important issue that can be raised is whether the weight change is transitory or if it can continue into adulthood.
Other major findings in the present study were behavioral changes induced by administration of fentanyl in our model. Somewhat surprisingly, the results point towards an overall apparently “positive” effect on neurodevelopment, instead of the expected negative one. This “positive” impact was evidenced by an apparent lack of significant acute toxic effects on early development. Moreover, later, in infant rats who were treated with fentanyl, we found enhancement of the recognition index of a novel object, lesser anxiety-like behavior, and better performances on the wire hanging maneuver and on the accelerating rotarod. Furthermore, there was a trend for sooner eye opening in this group, suggesting that the eye command center of CNS of rats in the fentanyl group ages earlier.
Interestingly, aversive stressful procedures performed in the current study, which should be associated with increased anxiety, seemed blunted by fentanyl treatment. In fact, fentanyl-treated rats were significantly less anxious than the saline and the unoperated rats in the elevated plus maze. This outcome is not clearly explained, but calmer subjects can probably better explain other outcomes found in this study, such as enhanced cognitive function, motor, and balance and coordination. It is possible that all these results are at least partially explained by a fentanyl impact on the development of central neuronal circuits, given the great plasticity of the CNS characteristic of the immature mammalian brain [23
The effects of the impact of fentanyl on SNC are probably complex and multivariate with different possible mechanisms found in the literature, both potentially protective or detrimental, such as faster CNS myelination and enhanced neurogenesis by NeuroD activity level increase (a transcription factor essential for the development of the CNS) [24
] eventually translating into enhanced performance or, on the other hand, cytotoxic lesion/blockade of the ventral hippocampus by N-methyl-D-aspartate (NMDA) receptor interference, manifesting as reduced anxiety [25
]. It is well known from the literature that fentanyl modulates important cellular and molecular neuronal mechanisms, interfering not only in anatomically distributed neural network involved in generating states of anesthesia but also in mechanisms involved in hippocampus neurogenesis. In this setting, fentanyl may regulate the functions of the developing hippocampus, a region highly related to learning, memory, stress responses, and emotionality [25
There is a growing body of evidence showing that drugs interfering in the SNC functions may cause pharmacologic neuroprotection or, on the opposite, detrimental effects, depending on the pathological conditions [19
]. Negative impact alerts are particularly alarming in the context of very ill preterm infants who usually present a multitude of physiological derangements and pathological pain conditions coupled with a very immature brain, therefore it is important to define safe indications and doses for the use of these drugs, such as fentanyl, in this stage.
In conclusion, the current study is the first to demonstrate that rat pups exposed to parenteral fentanyl in a painful context have lasting growth and behavioral changes. The study highlights behavioral changes that could potentially affect brain function either in a positive or negative manner. These results should serve as a basis for further research and should lead investigators to focus on specific pathways relevant to the changes in behavior we have shown. Our findings may contribute to support the neonatal use of fentanyl, when indicated, namely in postsurgical settings, even in premature newborns. However, extrapolating our data to a clinical setting must be done with caution, as with every animal study.