The impact of sex and obesity on traumatic brain injury outcomes : exploring the interplay between biological sex and high-fat diet in mice

<p dir="ltr">Traumatic brain injury (TBI) is a significant clinical problem with no effective therapeutic intervention available. The pathophysiology of TBI is highly heterogeneous and is known to be influenced by individual variability in genes and lifestyle. A better understanding of specific factors that can modulate TBI outcomes is necessary for the future development of effective treatment plans. A high-fat diet (HFD), with 40-60% kcal fat consumption, has been reported to increase inflammatory proteins, glial cell activation, and neuronal damage in the brain, which can potentially worsen TBI outcomes. Another potential factor that can affect TBI outcomes is sex. Female sex hormones, oestrogen and progesterone, have been found to exert neuroprotective effects after brain injury. The impact of HFD intake and sex difference on modulating neuroinflammation and behavioural outcomes after TBI have not been explored yet. To address this gap, this study was divided into 4 experiments to study the effects of both HFD and sex differences on neuronal function, neuroinflammation, and gut microbiome dysbiosis following TBI. <br>Together, the experiments described in this thesis aimed to determine whether HFD worsens TBI outcomes and whether the effect of TBI and/or diet are sex-dependent. To begin, the researcher conducted a pilot study, described in Chapter 3, to establish an obesity model that produces neuroinflammation and to optimise behaviour test protocols for use in subsequent studies. Three-week-old male mice (n = 12/group) were fed either HFD (45% kcal fat) or a standard chow diet (5.8% kcal fat). This study found that HFD consumption for 11 weeks had effectively induced obesity. The neurological testing commenced after an 11-week diet regimen, using several tests to assess motor function (vermicelli handling test, swim test, and open field), sensorimotor function (adhesive removal test), cognitive function (Y-maze short-term memory and Y-maze spontaneous alternation), and anxiety (open field and elevated plus maze). Although these behaviour tests are often used to detect impairment in experimental TBI studies, it is challenging to find behavioural tests that accurately detect impairment but do not rely on gravity movements, which could be impaired in heavier mice. Four out of seven behaviour tests were used in the subsequent experiment of Chapter 4, including swim tests, open field, adhesive removal test, and Y-maze spontaneous alternation, since both HFD and standard chow-fed mice could perform equally well in these behaviour tests. <br>Based on previous studies, the obesity model used in this study could increase inflammatory markers in the brain. To confirm if this obesity model could promote neuroinflammation, markers of activated astrocytes and microglia, including glial fibrillary acidic protein (GFAP) and ionised calcium-binding adaptor molecule 1 (Iba1), respectively, were quantified in brain areas, including the cortex, hippocampus, and hypothalamus. This study found that a HFD increased the expression of GFAP in the hypothalamus (n=6/group) but not in the cortex and hippocampal areas such as CA1, CA3, and dentate gyrus. No effect of HFD on Iba1 abundance was found in the cortex, hypothalamus, and hippocampus area (CA1, CA3, and dentate gyrus). The cytokine IL6 was quantified using ELISA. HFD consumption increased approximately 50% IL6 levels in the cortex; however, the IL6 levels in the hippocampus were similar between standard chow and HFD groups (n=5-6/group). Therefore, the effect of HFD on IL6 levels was investigated after TBI. To test this, mice (n = 3/group) were subjected to brain injury using the CCI model and euthanised 24 hours after brain injury. In this underpowered pilot study, HFD did not significantly affect the level of IL6 in the perilesional cortex and hippocampus; however, the levels were approximately 50% higher in the HFD cortex. Based on the preliminary findings of HFD-induced astrogliosis, this HFD formulation (45% kcal energy of fat) was used for the remaining experiments. <br>The experiment described in Chapter 4 aimed to investigate the effects of HFD and sex differences on behavioural outcomes following TBI. To address this aim, male and female C57Bl/6 mice were fed with HFD formulation (45% kcal energy of fat) and subjected to a moderate, unilateral focal TBI using controlled cortical impact or to sham surgery. Mice (n=10-13) were subjected to behavioural tasks post-surgery over two weeks, including open field (locomotor activity and anxiety), Y-maze spontaneous alternation (working memory function), swim test (motor function), and adhesive removal test (sensorimotor function). Data were analysed using a generalized linear model to assess the effects and the potential interactions of diet, sex, and injury on neurological function. In the open field test, TBI induced hyperactivity in females irrespective of diet, but only HFD male mice experienced hyperactivity after TBI. This study did not find any effect of sex, diet, and injury on working memory function. In the swim test, females were not affected by TBI, but males were, with the greatest deficit occurring in the HFD group. In the adhesive removal task, TBI caused sensorimotor function deficits in all sex and diet groups, and a HFD aggravated this effect in males. Overall, these results showed that diet and sex interact to affect outcomes following TBI, and these effects were different for different behavioural tasks. <br>To determine whether the relationship between sex and diet on behavioural outcomes following TBI could be associated with differences in the inflammatory response in these groups, the experiment in Chapter 5 aimed to investigate the effects and interactions of sex and diet on neuroinflammation following TBI. Cytokine and chemokines levels (IFNγ, TNFα, IL1β, IL6, KC, MCP1, and MIP1α) were measured in the pericontusional cortex and ipsilateral hippocampus at 24h post-surgery using a multiplex immunoassay (n=4-5/group). This results showed that IL6, KC, and MCP1 were higher post-TBI in females than in males with a chow diet. HFD decreased levels of these cytokines post-TBI in females while increasing MCP1 in males. In the ipsilateral hippocampus, TBI increased MCP1 and MIP1α expression across sex and diet, and HFD increased MIP1α expression in females. Activated astrocytes and microglia were also assessed to investigate the effect of HFD consumption and sex differences on cellular inflammation. TBI increased astrogliosis and microgliosis in all groups. However, there was no significant effect of HFD consumption or sex on the expression of astrocyte marker GFAP or the microglial marker Iba1 in the ipsilateral cortex. Healthy brain tissue volume was measured at 2 weeks post-TBI to assess the effect of HFD and sex on lesion volume progression after brain injury. Surprisingly, females had significantly lower healthy brain tissue volume than males following injury. Together, these findings suggest that neuroinflammation following TBI could be sex-dependent and influenced by HFD. A change in neuroinflammation may be associated with the effect of HFD and sex on behavioural outcomes following TBI; however, this study did not find an association between them. <br>The previous experiments found that HFD and sex differences affected TBI outcomes; however, the underlying mechanism remained unclear. Therefore, the following experiment, described in Chapter 6, investigated whether changes in the compositions of the gut microbiome could potentially underlie the effect of HFD and sex differences on TBI outcomes since previous studies showed that alternation of the gut microbiome could mediate neuroinflammation following TBI. This study assessed the changes of several gut bacteria in the presence of HFD, sex difference, and TBI in mice faecal matter by analysing the expression of five genera (<i>Prevotella, Bacteroides, Bifidobacterium, Enterococcus</i>, and <i>Lactobacillus</i>) and two species of gut microbiota (<i>Akkermansia municiphila</i>, and <i>Clostridium perferingens</i>) using qPCR. This study found the effect of sex and diet on <i>Bifidobacterium</i>; HFD increased the expression of <i>Akkermansia municiphila</i>; TBI decreased the expression of <i>Enterococcus spp</i>; and HFD decreased <i>Prevotella</i> after TBI in faecal matter. The remaining bacteria, <i>Bacteroides</i> and <i>Lactobacillus,</i> were not affected by sex, diet, or injury. These findings suggest that the effect of HFD on gut bacteria is sex-dependent and the gut microbiome changes may associated with TBI outcomes. <br>In summary, this study found that, overall, females performed better in behavioural tests than males, and a detrimental effect of HFD on behavioural outcomes following TBI was stronger in males than females. However, an effect of both sex and HFD was not found in the abundance of activated astrocytes and activated microglia. HFD did not affect healthy brain volume following TBI in both sexes; however, females showed significantly lower healthy brain volume than males. This finding supports the cytokines analysis results, in which females had higher KC, MCP1, and IL6 expression in the perilesional cortex. The effect of HFD differed between females and males. HFD decreased the expression of inflammatory protein levels in females but either had no effect or increased levels in males. A higher expression of <i>Bifidobacterium</i> in females than males suggested that females might be protected from the negative effect of HFD and TBI via beneficial alterations in the gut microbiome. Together, these findings extend the existing knowledge of the effect of sex differences and HFD-induced obesity on neuroinflammation, behavioural outcomes, and the gut microbiome following TBI.</p>