6. General discussion
6.3 What is new in our research on NACA
As far as we know, our study is the first study carried out on neuroprotective effects of NACA in neonatal pigs. We suggest that our findings of less expression of the inflammatory proteins IL-1 and phosphorylated NF-kB (p65) in the brain as well as TNF- in plasma, may indicate that NACA could play a role, alleviating the inflammatory process evolving after perinatal hypoxia-reoxygenation.
Further, the decreased levels of mtDNA in cerebellum in pigs subjected to NACA after the hypoxic challenge, along with the tendency to less proteolytic activity in the Purkinje cells, illustrated in article IV, could strengthen the theory that NACA has anti-inflammatory and neuroprotective properties after perinatal asphyxia.
Moreover, the promising results described in article III, dealing with the viability of embryonic kidney cells exposed to oxidative stress, point out that NACA may have protective effects in other organs in addition to cerebrum, when the neonate is exposed to oxidative stress. These articles add important
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information to our knowledge of NACA, as we are the first to describe its effects in a neonatal pig model as well as in an embryonic cell line.
It is worth mentioning that in a cell line of primary neurons, NACA was able to partly reverse the toxic effects of on the neurons, therefore NACA may reduce the degree of neurotoxicity induced by in the brains of AD patients suffering from AD [149] and we speculate that NACA could alleviate the development of AD in a number of patients.
6.4 Is there a link between perinatal asphyxia and AD
During the German occupation in the winter 1944-1945 the population of the western parts of the Netherland suffered from famine. The average intake of calories for pregnant women was only 400-800 kcal/day, about a quarter of the recommended amount of calories [202], and therefore many foetuses received less nutrients than recommended. This birth cohort, born in winter, spring or summer 1945, has later on been extensively investigated.
Studies on the Dutch Famine Birth Cohort, which consists of 2414 men and women from Amsterdam born around the time of the dutch famine, have revealed a link between prenatal conditions and typical diseases of aging [203, 204].
Even though several genes have been identified which contribute to AD, the most important being APP, Presenilin 1 and 2 and Apo4 [205], environmental factors seems to have an impact on AD. The Dutch Famine Birth cohort has a higher prevalence of some disorders associated with ageing. Individuals, who were in their early gestation during the famine, exhibited an increased risk of cardio-vascular and metabolic diseases more than 60 years later. Compared with persons who were born before the famine, they had significantly lower scores on cognitive tests [206]. These results are in line with other reports describing that intrauterine exposure to famine may lead to increased risk of diabetes, obesity, cardiovascular disease, schizophrenia and cognitive aging [203].
Therefore, it is not surprising if neonates exposed to severe hypoxia at birth could be more prone to adult neurodegenerative diseases such as AD.
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6.5 What is new in our research on -Amyloid
To our knowledge, we are the first who have investigated the biomarkers Tau and A42 in a neonatal population, and the reduction of A42 in CSF after perinatal asphyxia has not been previously described.
The function of A and its precursor APP are not fully elucidated. B-APP knockout mice display severe cognitive defects, indicating that A may have important functions in the nervous system [207].
However, the protein A is able to misfold and aggregate into oligomers and these oligomers are thought to have a toxic influence on the neurons in AD [208]. Lambert et al. showed in a rodent model that small diffusible A
oligomers could be fatal to mature neurons in hippocampus [209]. Further, Pillot et al. were able to demonstrate that small non-fibrillar complexes of A
could disturb the plasma membrane of neurons and induce toxicity in primary culture from cortical neurons via an apoptotic pathway [210, 211]. The theory that humans subjected to perinatal asphyxia are more susceptible to AD in late adulthood is further strengthened by the findings of Bernert et al. who demonstrated that neurodegeneration and neurotransmitter changes were present in adult guinea pig 3 months after exposure to perinatal asphyxia [212].
Several papers have described a possible association between traumatic head injury and AD [23] and Magnoni et al. showed that the level of Ain the extracellular space was increased after head trauma.
[27]. Furthermore, a leakage of S100B from the astrocytes and an elevation of S100B in CSF and plasma evolve after traumatic brain damage [213, 214].
The above mentioned findings, regarding high levels of S100B in CSF and plasma after head injury and decreased levels of Ain the extracellular space after trauma to the head are comparable with our study, where we demonstrated a decrease of A42 combined with an increase of CSF- and plasma-S100B. It is tempting to speculate that the lower levels of A42 in the pigs exposed to hypoxia could be a sign of aggregation of A42, which in turn attacks the neurons, triggering a long-lasting process, which makes them more susceptible to AD in late adulthood, when the organism is more exposed to ROS. The absent changes of the biomarkers T-tau and p-Tau could be due
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to the limited time of our experiments, 9.5 hours, and we suggest that this time frame is too short to develop tau changes.
Individuals who are in their 60´ies and 70´ies today, the age when the first signs of AD are usually presented, were born at a time when no systematic assessment for asphyxia, such as Apgar or blood gases, were performed.
From 1969 onwards, Apgar score has been measured in all Norwegian children. Therefore, it would be highly interesting to conduct a register-based study from 2030 onwards, to compare if neonates scored with a low Apgar score, show more signs of AD than their peers.
7. Considerations
Quantifying the expression and activation of genes and proteins at one time-point does not reflect the whole pathophysiological process after perinatal asphyxia. Further, one may argue that more pigs should be included to increase the statistical power, however; in our research a higher number of animals were investigated than in many comparable studies.
Finding the right dose of an antioxidant may be a time-consuming and difficult task as several antioxidants may be chemically modified to pro-oxidants when administered in higher doses [137].
Estrella et al. showed three decades ago that Acetylcysteine may have pro-oxidant properties and the degree of pro- versus antipro-oxidant effects was dependant on the administered dose [138]. Therefore, we cannot exclude the possibility that even NACA may have pro-oxidant abilities when given in high doses.
7.1 Implications for further research
Our work may implicate that NACA have more effects regarding neuroprotection than previously known. However, more studies, including different concentrations of NACA, must be conducted before NACA should be considered used in a clinical trial. One important question which should be answered before initiating such a trial is if NACA, like N-Acetylcysteine, may initiate release of Histamine [135] and cause side effects such as anaphylaxis and hypotension [215]. In future studies, assessing the effects of NACA after
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perinatal asphyxia, hypothermia should be inflicted to the animals, because hypothermia is now a well established prescribed procedure started after hypoxia-reoxygenation in the newborn [216, 217].
8. Summary
In our paper addressing the question whether NACA may reduce the mortality in a porcine epithelial-like embryonic EFN-R kidney cell line exposed to H2O2,
we demonstrate that NACA may reduce cell death after exposure to oxidative stress. Further, we present that the levels of Il-1 and NF-kB in cortex were lower in pigs exposed to NACA after hypoxia compared to pigs exposed to saline, which may indicate that NACA may reduce signs of cerebral inflammation. However, of the cytokines investigated, only a few were influenced of the administration of NACA,
In cerebellum we found that NACA may reduce signs of proteolytic avtivity and mutations of mtDNA which may indicate that NACA may exhibit some neuroprotective effects.
Due to the findings of the reduced concentration of A42 combined with the increased levels of S100B in the CSF of the NACA-pigs, we suggest that biomarkers of adult neurodegenerative diseases may play a role in the evaluation of HIE.
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