Abstract
Schizophrenia is a devastating life-long psychiatric disorder with manifestation in late adolescence or early adulthood. Epidemiological research shows that prenatal exposure to viral or bacterial infection during the second trimester of gestation increases the likelihood of the offspring developing schizophrenia in adulthood. The maternal immune activation (MIA) rodent model has therefore been widely used to address the neurodevelopmental aspect of schizophrenia. MIA offspring display a variety of structural, morphological and neurochemical changes in the brain and behavioural alterations that resemble schizophrenic patients. Dysfunction of microglia, the innate immune cells in the brain, has also been linked to the etiology of schizophrenia.
L-arginine, a semi-essential amino acid, is nutritionally essential for the neurodevelopment. It can be metabolised by nitric oxide synthase (NOS), arginase and arginine decarboxylase to produce a number of bioactive metabolites. Recent research has implicated altered arginine metabolism in the pathogenesis of schizophrenia. MIA induced by the viral mimic polyinosinic-polycytidylic acid (PolyI:C) on gestational day (GD) 15 affects brain arginine metabolism in the adult and postnatal day (PND) 2 neonatal rat offspring. Intriguingly, immunohistochemistry revealed increased intensity of neuronal NOS (nNOS)-positive cells and disrupted microglial migration and maturation in PND2 MIA offspring. It is unclear, however, whether these changes are transient or persistent in MIA rat offspring. A time-course study was therefore designed to systematically investigate how a single MIA insult induced by PolyI:C affected arginine metabolic profile in the brain and blood, nNOS and microglia immunoreactivity in the hippocampus and behavioural functions in male and female offspring at the age points of PND 7, 14, 21, 35 and 60 (Experiments 2-5).
Since both male and female offspring were used, Experiment 1 determined the effects of sex and estrous cycle on arginine metabolic profile in the brain and blood (plasma) using adult rats. Female rats displayed significantly lower levels of L-arginine in the frontal cortex and three sub-regions of the hippocampus when compared to male rats. Moreover, female rats had significantly higher levels of L-arginine and γ-aminobutyric acid, but lower levels of L-ornithine, agmatine and putrescine, in plasma relative to male rats. The observed sex difference in brain L-arginine appeared to be independent of the enzymes involved in its metabolism, de novo synthesis and blood-to-brain transport (cationic acid transporter 1 protein expression at least), as well as circulating L-arginine. While the estrous cycle did not affect L-arginine and its metabolites in the brain, there were estrous cycle phase-dependent changes in plasma L-arginine. These findings, for the first time, demonstrate the sex difference in brain L-arginine in the estrous cycle-independent manner. Since peripheral blood has been increasingly used to identify biomarkers of brain pathology, the influences of sex and estrous cycle on blood arginine metabolic profile need attention when experimental research involves female rodents.
Experiment 2 set up and validated the MIA rat model induced by a single tail vein injection of PolyI:C on GD15. One pair of male and female control or MIA offspring from each litter were assigned into each of the five age groups (PND7, 14, 21, 35 and 60) for Experiments 3-5. PolyI:C resulted in a marked body weight loss in pregnant rats on the day of the injection, indicating the efficacy of its administration. Male and female offspring at PND35 and 60 displayed the deficits of prepulse inhibition (PPI), a hallmark feature of animal models of schizophrenia, as well as impaired behavioural performance in the open field and Y-maze, relative to their age- and sex-matched controls. Collectively, these results confirmed the successful set up of the MIA rat model for the following three experiments.
Experiment 3 systematically investigated the effects of MIA on arginine metabolic profile in the brain and plasma of offspring at PND 7-60. Regarding the brain profile, MIA offspring displayed reduced NOS activity and nNOS expression but increased level of agmatine at PND7, and increased NOS activity, nNOS and/or inducible NOS (iNOS) expression and glutamine/glutamate ratio but reduced agmatine levels at later age points (PND 35 and 60 in particular). Regarding the plasma profile, there were reduced agmatine levels and increased glutamine/glutamate ratio in MIA offspring at PND 21, 35 and 60. The receiver-operator characteristic curves revealed the possibility of plasma agmatine in detecting male MIA offspring at PND35. These findings further demonstrate that a single MIA insult alters brain and blood arginine metabolic profiles in offspring even at the early postnatal age, although more changes are evident in adolescent and young adult offspring. Moreover, there were sex-dependent changes in MIA offspring in the age- and/or region-specific manner.
Via a stereology approach, immunohistochemistry was performed to determine the effects of MIA on hippocampal nNOS immunoreactive (nNOS-IR) cells and microglia in offspring at PND 7-60. Experiment 4 found reduced number and density of nNOS-IR cells in the CA1 and CA2/3 sub-regions of dorsal hippocampus in male and female MIA offspring at PND7, but not other age points, further suggesting a transient reduction in nNOS expression in the hippocampus. There were also sex differences in nNOS-IR cells in the dorsal hippocampus in offspring at PND14 and PND35 regardless of treatment. Experiment 5 observed the early and persistent reduction in the total number and density of microglia accompanied with altered morphology in the CA1, CA2/3 and DG sub-regions of the dorsal hippocampus in both male and female MIA offspring at all five age points. Moreover, there were sex differences in microglia and sex-specific MIA effects in the age- and region-specific manner. Given the roles of microglia in synaptic maturation and brain wiring during neurodevelopment, MIA-induced microglial maldevelopment and/or dysfunction would have a dramatic impact on the brain functions of offspring.
The present study, for the first time, demonstrated that a single MIA insult induced by PolyI:C on GD 15 resulted in behavioural deficits, and dynamic or persistent changes in brain and plasma arginine metabolic profiles and hippocampal microglia in both male and female rat offspring at PND 7-60. These results further support the significant impact of MIA on offspring. The ongoing Covid-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 200 million infections worldwide, including pregnant women. Given the well-known link between MIA and offspring neuropsychiatric illness risk, the impact of SARS-CoV-2 infection on pregnant women and consequently their offspring will be a critical health issue worldwide. Since the immune response SARS-CoV-2 initiates bears some similarities to that of PolyI:C, the results obtained from this project may enhance the understanding of the impact of SARS-CoV-2-induced MIA on offspring and assist the development of the preventive/therapeutic strategies for SARS-CoV-2 infected offspring.