Abstract
Infants born extremely premature have a significant risk of experiencing repeated hypoxic (RH) insults. RH insults in extremely premature infants, due to underdeveloped control of breathing, is associated with increased production of free radicals. Oxidative stress and glutamate toxicity from free radicals can lead to neuropathologies. The most common behavioural disorder associated with extreme prematurity is attention deficit hyperactivity disorder (ADHD). Extremely premature children also have a higher risk of having auditory neuropathy spectrum disorder (ANSD). ANSD accounts for about 10% to 15% of permanent childhood hearing loss. A prior study conducted using the same animal model showed a significant decrease in the total number of neurons in one region of the inferior colliculus (IC), a key structure in the auditory pathway, of repeated hypoxic rats in comparison to normoxic rats. Melatonin is a potent antioxidant (i.e. free radical scavenger) and anti-inflammatory naturally produced in humans, with a longer period of intrinsic deficiency evident in extremely premature infants. Thereby, melatonin has the potential to be a conservative, early onset neuroprotective treatment for brain injury caused by extreme prematurity. Caffeine and other forms of methylxanthines have been used since the 1970s and have had extensive research suggesting better neurological outcomes. We tested the hypothesis that melatonin or caffeine can rescue the neurons in the left IC in an extremely premature hypoxic rat model of the hyperactive, impulsive and attentive subtype of ADHD. The left IC was investigated because the right side of the brain was used in earlier research to investigate the midbrain dopaminergic system and ADHD. ADHD is a right brain disorder. Male Sprague Dawley rats at post-natal day (PN) 1 – 3 were subjected to repeated sublethal doses of hypoxia every 2 hours, between 7am and 7pm, for 12 hours a day. One of the three treatments (i.e. Diluent, melatonin or caffeine) were administered between PN1 – 3. Melatonin (10 mg/kg, n = 10 rats) was administered subcutaneously immediately after the second hypoxic exposure on PN1, before the first hypoxic exposure on PN2 and PN3, and after the last hypoxic exposure of each day between PN1 – 3. Caffeine (n = 10 rats) was administered subcutaneously immediately after the second hypoxic exposure on PN1 and before the first hypoxic exposure on PN2 and PN3. The dose on PN1 was 10 mg/kg and 2.5 mg/kg on PN2 and PN3. Diluent (n = 9 rats) were subcutaneously injected with a vehicle solution of 2% ethanol in saline following the administration schedule of the melatonin treatment. The rats treated with melatonin showed a statistically significant increased sitting time at 5pm on PN3, suggesting that the brain was exposed to exogenous melatonin, including the IC. The rats treated with caffeine showed an increased mortality rate at PN3, in a previous experiment using a higher dose over PN1 – 3, indicating that the caffeine passed through the blood-brain barrier and affected the brain. At PN210-PN213, the animals were perfused and the left IC of n = 8 animals per group were serially sectioned in the coronal plane using 40 µm frozen sections. The serial sections were then stained with cresyl violet. The three subdivisions of the IC, the external cortex (ECIC), central nucleus (CIC) and dorsal cortex (DCIC), were then stereologically analysed using the Cavalieri’s and optical disector methods. There was no statistically significant difference between the three treatment groups in the: body weight, absolute volume of the left ECIC, CIC, DCIC and IC, average number of neurons in a sub-volume, and the absolute number of neurons in the left IC and its three subdivisions. This study suggests that melatonin (at 10 mg/kg during PN 1 – 3) and caffeine (10 mg/kg at PN1 and 2.5 mg/kg on PN2 and PN3) does not rescue the neurons in the left IC in a RH rat model of extreme prematurity.