A thyroid hormone based therapy to restore brain maturation following foetal growth restriction

Intrauterine growth restriction (IUGR) is a condition in which a foetus does not reach its full genetic growth potential, and is a leading cause of perinatal death and postnatal morbidity. IUGR often results in permanent neurodevelopmental deficits, ranging from cognitive and behavioural impairments to cerebral palsy. IUGR can occur due to a number of environmental or genetic factors, however the dominant cause is a lack of oxygen and nutrients delivered the foetus as a result of placental insufficiency. Deficits in grey matter (GM) and white matter (WM) in the foetal brain are thought to underlie these neurodevelopmental sequelae. There is no current treatment to prevent or correct placental insufficiency, and babies are often delivered preterm if IUGR is found to be severe or worsening, further increasing the potential for adverse outcomes. With 27,000 IUGR babies born in Australia in the past year alone, and 30 million born worldwide, new therapies which can be delivered immediately after birth, are urgently required.

IUGR decreases the expression of monocarboxylate transporter-8 (MCT8) (Azhan, 2019, Chan et al., 2014), a transporter protein necessary for the delivery of thyroid hormone (TH) into cells such as oligodendrocytes (OLs) in the brain (Lee et al., 2017). TH is critical for the maturation of OLs during foetal brain development, and reduced transport of TH into OLs impairs myelination (Lee et al., 2017). In this thesis, it is proposed that the synthetic TH analogue 3,5-diiodothyropropionic acid (DITPA) which does not require MCT8 to enter cells, can be used to overcome deficits caused by a loss of MCT8 expression in the IUGR brain, thereby restoring myelination. Our group has previously shown that MCT8 mRNA levels are reduced in the newborn IUGR rat at postnatal day (P) P7, and these levels normalise by P14 (Azhan, A., PhD thesis, Monash University, 2019). Furthermore, we have previously shown that short-term administration of DITPA (0.5mg/100g/day i.p.) to newborn IUGR rats (from P1 to P6) corrected the myelination deficit in the external capsule by P7, and did not affect neonatal growth parameters (Azhan, A., PhD thesis, Monash University, 2019). The present project set out to investigate the benefits of a longer-term DITPA treatment from P1 to P13 (0.5mg/100g/day i.p.) a time in rat brain development equivalent to 23 to 40 weeks gestation in the human (Semple et al., 2013), and reflective of what is likely to occur in the clinical setting with an IUGR baby delivered preterm and given DITPA until term equivalent age. The same cohort of animals contributed to all 3 experimental chapters in this thesis.

In Chapter 3, the impact of DITPA administration on the development of the cerebrum was investigated, focusing on myelination, a neurodevelopmental process affected in IUGR, and regions with known vulnerability to the prenatal insults. Specifically, this study aimed to determine whether DITPA administration in IUGR rat pups from P1 to P13 improved myelination, promoted OL maturation, and did not cause injury or inflammation in the cerebrum compared to vehicle (saline) treatment. The data presented in Chapter 3 shows that DITPA treatment in IUGR may promote myelination in the cerebral cortex and fimbria, increase OL density in the corpus callosum, and does not cause injury or inflammation in the cortex, corpus callosum, hippocampus or fimbria when assessed at P14. This study indicates that an extended duration of DITPA treatment may be beneficial to myelination in the IUGR cerebrum but is not favourable when given in controls, decreasing myelin proteolipid protein (PLP) and OLs the cortex and fimbria. Next, it was essential to investigate DITPA's therapeutic potential in another brain region with known vulnerability to IUGR (De Bie et al., 2011, Padilla et al., 2011), the cerebellum.

The study presented in Chapter 4 aimed to determine the impact of DITPA treatment (P1 to P13) on myelination, OL development, morphology, and neuroinflammation in the cerebellum in IUGR rat pups. DITPA did not improve myelination or promote OL maturation in the cerebellum of IUGR pups when assessed at P14, had no negative impact on cerebellar morphology (layer widths/areas and Bergmann glial fibre density), however increased the density of Purkinje cells, and microglia in late developing cerebellar lobules. Overall, these data support longer-term DITPA administration in IUGR as exhibiting more benefit in the cerebrum than the cerebellum; as in the cerebrum DITPA may be unfavourable when given to controls. To further investigate the potential use of DITPA in treating IUGR, potential off-target effects on neonatal growth and wellbeing were determined next.

Chapter 5 examined potential off-target effects of DITPA administration in control and IUGR neonates, focussing on neonatal growth, and assessment of wellbeing. Body weight of all pups was assessed at P1, P7 and P14. Liver and kidney weights, body morphometry (head and hip circumference and crown-to-rump length), body composition (bone density and mineral content, lean tissue mass, fat mass), thyroid and liver function, as well as cholesterol levels were measure in pups at post-mortem (P14). DITPA administration in IUGR pups did not adversely impact neonatal growth, brain or organ weights or body composition, despite altering FT4 levels and showing hepatic thyromimetic activity, but alters growth, liver weight and bone mineral content when given to control pups.

In conclusion, this thesis has, for the first time, demonstrated that longer-term administration of the TH analogue DITPA, which enters cells independently of MCT8, to newborn IUGR rats promotes myelination in the cerebrum, increases OL density in the corpus callosum, and does not cause injury or inflammation to the brain, albeit for a possible inflammatory response in the late developing cerebellar lobules. In IUGR pups there were no negative off-target effects on neonatal growth or wellbeing, although DITPA caused unfavourable outcomes when administered to controls. The work presented in this thesis collectively highlights the potential for DITPA to improve myelination outcomes in the IUGR brain, without causing injury or adverse off-target physiological effects, however further studies are required before DITPA can be considered as a therapy in IUGR.