title: Investigating opiate-induced changes on respiratory pattern and brain blood flow in mice
Creators: name: Damanhori, Majdi Mohammedzaki
Publisher: name: University of Glasgow
Year: 2022

description: Breathing is a rhythmic motor process thatstarts at the perinatal age, influenced by other physiological measures, and continues throughout the life course of a mammal. Failure to produce arespiratory rhythm can be fatalandunderstanding the mechanism of the respiratory rhythm generation by the brainstem is crucial. In vitro and in vivo studies have disclosed evidence ofa region within the ventrolateral medulla, known as the pre-Bötzinger complex (preBötC), and assumes it to be the respiratory rhythmogenesis originator. Also, some studies suggest the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) as a second respiratory rhythm generator that plays a certain role in in the neonatal rodent. At neonatal age in rodents and humans, thebreathing pattern is irregular and respiratory system is immature, which signifies that it isvulnerable to external environmental challengesduring this period. Nevertheless, the maturity step occurs early in life and breathing becomes stable and constant. The mechanisms of respiratory rhythm generation perinatally remains elusive, and it is hypothesised that the RTN/pFRG acts as the dominant respiratory rhythm generator during the early days of life when the respiratory system is immature. After that, preBötC takesthe role of the rhythm generation, and breathing becomes more stable. However, the interactionbetween preBötC and RTN/pFRG in vivo during postnatal development is not fully understood. Fentanyl is widely used on a clinical basis to control chronic and acute pain. Despite the fact that respiratory depression is a common side effectof fentanyl, the long-term respiratory consequences of repeated fentanyl exposure is unknown. Concerning the immaturity of the respiratory system during neonatal life in mammals, the thesis was designed to understand the long-term effects of fentanyl exposure during this vulnerable period in neonatal rodents, which is a suitable model for understanding long-lasting changes in humans. Therefore, the fentanyl-exposure at postnatal stage was categorised into two groups, which are neonatal mice (NN) exposed to fentanyl (0.04 mg/kg daily) from P1-P5 and juvenile mice (JUV) exposed from P9-P13. The first aim of the thesis was to investigate the influence of fentanyl on the postnatal maturation step of the central respiratory control. Fentanyl, a potent μopioid receptor agonist, was introduced to evoke respiratory frequency depression in vivo by targeting preBötC neurons, which aresensitivetoμ opioids, while the RTN/pFRG neurons are insensitive to μ opioids. Therefore, fentanyl was used to suppress the preBötC in vivo throughout the postnatal developmental phase. Then, the effect of the early fentanyl exposure was evaluated duringadulthood via plethysmography. The plethysmography data showeddifferences in the respiratory rateat restbetween the NN and the control group. Also, the awake fentanyl challenge showed an increase inthe tidal volumeresponse to fentanyl in the NN group. The fentanyl challenge was applied to the study groups to evaluate the vulnerability and sensitivity to fentanyl under anaesthesia. The vulnerability to fentanyl was observed in the NN and JUV groups, which shows lowsurvival rates of the fentanyl challenge. This indicates that the anaesthetised state is more sensitive to respiratory depression. Also, cerebral blood flow (CBF) was analysed during the same challenge to investigate the effect of opioids on CBF. An increase in the CBF was recorded via the laser speckle technique as a response to opioid exposure, and the fentanyl-exposed groups (NN and JUV) showed an attenuated response to opioid exposure within the parietal regions. The second aim of the thesis was to highlight the chronic effect of early-life fentanyl exposure on endogenous opioids and stress hormones by utilising the enzyme-linked immunosorbent assay (ELISA) technique. The study showed low detection of the endogenous opioid level (b-endorphin) due to the small blood volume used for the analysis. Yet, the JUV-FEN group had more detectable levels of endogenous opioids, which could support the effect of earlier opioids exposure on endogenous opioids level. Weighted average trends were obtained in the JUV-FEN group compared to JUV-SAL, which were not statistically significant. In addition, a trend of the stress hormone (corticosterone) collected from hair follicle samples was noted in the JUV-FEN group. The third aim of the thesis was to investigate the long-lasting effect of early-life fentanyl exposure on the μ opioid receptor (μOR) neural expression and distribution within the CNS. Unfortunately, the initial group had cross-contamination on the films, and the samples were discarded. Therefore, other groups were involved in the study that showed a slight decrease of μOR density across the brain regions in the JUV-FEN group compared to JUV-SAL. Nevertheless, the presented data were underpowered regarding the small sample size involved in this other group.The last study of μOR neural expression was planned to be investigated via Immunohistochemistry (IHC) for brainstem and cerebral regions. Still, due to the national lockdown and the pandemic situation, it was not completed as planned. In conclusion, repeated early-life exposure to fentanyl possibly leads to long-lasting changes thatmayaffect the breathing pattern at rest and last into adulthood. Those changes are attenuated under anaesthetic and may lead to respiratory arrest.