Montelukast reduces grey matter abnormalities and functional deficits in a mouse model of inflammation-induced encephalopathy of prematurity

Preterm birth occurs in 11% of live births worldwide [1, 2], with 30% of the surviving population suffering from life-long neurological deficits [3‐5]. The varied neuropathology, collectively termed encephalopathy of prematurity (EoP), includes grey and white matter injury and increases the incidence of a range of cognitive and motor deficits in later life [6], for which limited treatments are currently available.

Whilst white matter (WM) injury has been the historical focus of preterm brain injury studies, there is increasing evidence of the importance for grey matter (GM) injury in this condition [7]. Neuropathological studies in clinical cohorts, and animal models of preterm injury, have identified decreased neuronal number [8, 9]; dendritic arborisation [10] neurogenesis [11], and developmental alterations in interneuron populations [12, 13]. Clinical magnetic resonance image (MRI) studies have also identified atypical GM development, including smaller cortical volume in EoP [14‐16]. Reduced cortical growth from 23 to 45 postmenstrual age (PMA), as assessed by MRI, predicted cognitive deficits at 2 and 6 years of age in preterm-born infants [17]. Diffusion MRI (dMRI) studies have also shown alterations in cortical microstructure assessed with diffusion tensor imaging (DTI) metrics [18, 19], suggesting a potential reorganisation of the developing neurons associated with preterm birth [20].

The underlying causes of EoP are multifactorial and most likely interlinked, with intrauterine growth restriction [21‐23], inflammation [24, 25], and chronic hypoxia [26‐28] recapitulating aspects of the neuropathology in animal models, and associated with clinical EoP. Microglial activation is well-established in the preterm brain with EoP [29, 30], and long-term epidemiological studies have shown that prolonged systemic elevation of pro-inflammatory cytokines correlates with altered brain development and poor cognitive outcome [24, 31]. Mediators of inflammation regulate many normal processes of brain development (reviewed by [32]) and experimentally induced maternal or systemic inflammation (produced with a variety of inflammatory mediators, including IL-1β) can recapitulate the above mentioned pathological processes of EoP; i.e. activate microglia, reduce neurogenesis, delay myelination, alter neuronal development and cause memory impairment [8, 13, 33‐36]. However, to date, therapeutic targeting of either inflammatory or other mechanisms of injury has not been translated to the clinic [37, 38].

The cysteinyl leukotrienes (CysLTs) are a family of highly potent pro-inflammatory mediators, derived from arachidonic acid via the 5-lipoxygenase pathway. They have been associated with a range of neuroinflammatory diseases including cerebral ischaemia [39, 40], Alzheimer’s disease [41], multiple sclerosis [42] and traumatic brain injury [43]. Leukotrienes act through the CysLT receptors (CysLTR), G protein-coupled receptors which are widely expressed throughout the body, particularly on immune cells, and in immune and contractile tissue [44]. In the central nervous system (CNS), the CysLTR1 antagonist, montelukast, has been found to increase neurogenesis [45], reduce microglial activation [46] and blood–brain barrier permeability [47], and support oligodendrocyte development [48], in addition to its immediate anti-inflammatory properties [49]. This data implies that montelukast treatment could ameliorate key pathologies identified in the developing brain subsequent of systemic inflammation [33, 50‐52]. Moreover, it has been shown to reduce infarct volume, demyelination, and seizure frequency in adult rodent models of neurological injury [47, 53‐56]. Importantly, CysLTs and their primary receptors are not constitutively expressed in the brain [55, 57], and therefore are unlikely to contribute to the neuropoietic cytokine signalling that regulates normal brain development, but their expression is upregulated following injury [53, 58, 59]. As such, we have identified montelukast as a potential therapy for preterm brain injury.

Montelukast is an attractive candidate for repurposing; it is currently utilised as an asthma medication; it has a good safety profile and is approved for administration to infants from 6 months of age. In addition, studies have shown that it is safe and that there are predictable changes in its pharmacokinetics in 0- to 6-month-old infants [60, 61]. Here, we show that concomitant treatment with montelukast reduces central and peripheral inflammation, long-term behaviour deficits and aspects of the underlying GM neuropathology related to EoP.

Preterm brain injury causes life-long cognitive, motor and behavioural deficits. Most studies have focused on WM injury, and, to date, no treatments have progressed to the clinic [38]. Recently, GM injury has been recognised as an important primary injury in EoP, which opens the scope for therapeutic development. Data presented in this study show improvement in GM deficits in a model of preterm brain injury following treatment with montelukast, a cysteinyl leukotriene receptor antagonist. Specifically, in this inflammatory model of mild perinatal brain injury, montelukast reduced the precipitating inflammatory event and attenuated the interneuronopathy. Furthermore, the increased MD detected in the adult cortex with injury was not detected in the animals that receive montelukast concomitant with the inflammatory injury. In turn, montelukast was able to normalise behavioural deficits, suggesting a functional link between GM deficits and some behavioural outcomes in preterm infants. We therefore propose montelukast as a potential therapy to ameliorate GM injury and associated behavioural deficits.

Montelukast was specifically chosen as a potential therapeutic agent for this condition as, in addition to being widely used and safe in children, it regulates a branch of the inflammatory response not constitutively active within the brain. Neuropoietic cytokines are important for providing a number of proliferation, differentiation and migration signals in the developing brain (reviewed by [32, 87]), which are fundamental for normal brain development. The CystLR1 and CystLR2 GPCR receptors are not constitutively expressed in the brain but are upregulated with injury [58, 59], allowing regulation of injury without affecting normal developmental processes. Of note, the G protein-coupled orphan receptor GPR17 has recently been identified as contributing to oligodendrocyte and myelination [48, 88], but understanding of its role in injury is limited.

Montelukast was successful at ameliorating the established central and, to a lesser degree, peripheral pro-inflammatory response. It is still unclear how inflammation is specifically signalled from the peripheral to the central compartment of the body in this perinatal injury model, and whether montelukast exerts its effects directly on the brain. There were small but significant increases in the expression of CysLTR1, the receptor target for montelukast, and CysLT concentrations in the brain following systemic inflammation that suggest montelukast could have its actions centrally, should it be able to access the receptors. Age and brain region-specific alterations in blood–brain barrier permeability have previously been reported in models of perinatal brain injury [50, 89, 90], though chronic systemic inflammation has previously been shown to induce central inflammation irrespective of blood–brain barrier function [91]. Here, we found no significant alteration in blood–brain barrier permeability, suggesting inflammatory mediators cross the intact barrier or generate central inflammation following secretion from the cerebrovasculature [92]. Montelukast may modulate this process through either peripheral or central actions. Montelukast was considered to be excluded from the brain (FDA, montelukast, Singulair®, application number 20-829), though a limited, but still significant entry to the brain has been reported that could explain its neuroprotective effects [93]. Consistent with this, in the present study, montelukast was detected in the brain, further supporting the possibility of a direct effect. However, the amount of montelukast detected was a small fraction of that in the plasma, and the upregulation of the CysLTR1 is also small, reducing the likelihood that the primary action is direct to the CNS. Based on these data, we hypothesis that the primary mechanisms for action of montelukast in this mouse model is to modulate the signalling of inflammation from the periphery to the brain, mostly likely at the blood–brain barrier. The greater effect of montelukast on the central, rather than peripheral, inflammatory response, and the relatively low production of leukotrienes and entry of montelukast to the brain support this hypothesis. Reducing the inflammation signalled to the brain will result in downstream therapeutic benefits in the CNS resulting from less central inflammation, and the reduction of short- and long-term neuropathology suggest a prevention or amelioration of some aspects of the injury, rather than a regenerative response. Further work is required to test this hypothesis, and to determine the clinical situations in which this preventative approach is appropriate. As the amelioration of the peripheral response was not complete, we also hypothesise that adjunct anti-inflammatory therapy could be advantageous in a future clinical treatment regimen. However, central inflammation, the key driver of injury in this model, was substantially reduced as early as 4 h after injury induction, which likely contributes to preventing the later decreases in parvalbumin interneuron number and behavioural deficits.

Injury to cortical parvalbumin interneurons has previously been described in this model [13], a cell type that has been shown to be selectively sensitive to inflammation [94]. Parvalbumin interneurons are the largest population of interneurons in the cortex and play an important role in integrating excitatory and inhibitory neuronal signalling. Altered parvalbumin function in the brain has been associated with a range of behavioural deficits, including memory dysfunction and impaired social interaction, repetitive behaviours, and increased seizure risk [95, 96]. Critically, the number of parvalbumin interneurons was normalised by montelukast as early as P10, indicating that early treatment can prevent GM injury caused by inflammation-associated preterm brain injury.

Similarly, an increased MD in the cortex in the inflammatory-injury group was not detected with montelukast treatment. Interestingly, a decrease in FA was observed only with a combination of inflammatory injury and montelukast treatment, compared to saline-treated controls. Assessments of normal early human [18, 97] and rodent cortical development [98] show that FA and MD decrease with increased cellular complexity, cell number and arborisation. Consistently, studies have shown increased MD as a result of cell loss [98, 99] or reduced arborisation [10]. In the present study, we have seen no evidence of extensive cell loss, and our previous studies using this model have not shown changes in cortical arborisation [13]. It is likely that more long-term neuropathological changes exist than have been identified in this study, as the differences observed with diffusion MRI metrics in the cortex between treatment groups are not easily explained by the histological findings of this study. Of note, MD can detect loss of a relatively small proportion of cells, and it has also been hypothesised that reduced size of myelinated axons can be detected with this metric [99]. This may explain both why long-term neuropathological correlates of this injury have not been detected to date and the slight (non-significant) decrease in FA in the cortex of the inflammatory-injury group. Petrenko et al. [99] also suggest some compensatory increase in arborisation of remaining cells after low level cell loss. It is, therefore, also possible that the FA result in our study reflects an undetected increase in dendritic arborisation driven by a combined action of inflammatory injury and montelukast treatment.

Montelukast was able to protect from inflammation-induced behavioural deficits in a mouse model of EoP. Improvements in anxiety and spatial learning were evident, which coexisted with the normalisation of parvalbumin interneurons early in development, and long-term organisation of cortical myelinated fibres as well as absence of the inflammation-induced increase in MD. Collectively, these findings suggest that GM injury may play a greater role in EoP than previously appreciated. Certainly, deficits in parvalbumin interneurons have been associated with these specific behaviour changes [94, 100]. In preterm infants, changes in cortical volume, cortical folding and microstructural connectivity have all been reported, which correlated with poor behavioural outcomes [15, 17, 101]. This suggests that subtle, early injury, such as the transient and selective parvalbumin interneuronopathy seen here, may be important factors in influencing outcome, and essential targets for therapy.

Work remains to determine the clinical situations in which this preventative approach is most appropriate, as some of the inflammatory drive may start prior to birth. Our hypothesis suggests that preventing inflammatory signalling from the periphery to the brain can limit down stream injury but does require therapeutic targeting of this inflammatory response early in the disease process. This can be tested in our preclinical mouse model and should be considered for meaningful evaluation of montelukast’s potential in future clinical trials. Of note, this preventative/ameliorative action was not seen on the early white matter injury, suggesting either an increased susceptibility of myelin to inflammatory injury that was not completely ameliorated or an interaction between montelukast and the developing oligodendrocytes that warrants further investigation. While this hypothesised mechanism is of scientific interest, and will be investigated, the key finding of ameliorated neuropathology is clearly evidence of the potential utility of this therapeutic approach. Future studies should also consider the broader beneficial potential of montelukast of other inflammation-associated pathologies in the preterm infant [102].

In conclusion, this study has demonstrated the importance of GM injury in EoP and that montelukast is a safe, repurposable drug. Montelukast is an effective treatment at reducing central inflammation, normalising parvalbumin interneuron numbers, and behavioural deficits associated with EoP in an inflammatory model of preterm brain injury. We therefore propose that montelukast has potential as a novel therapy for preterm brain injury.