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Investigating the therapeutic efficacy of non-anticoagulant fractions (Dp2 and Dp4) of enoxaparin following experimental traumatic brain injury

Aiyede, ME ORCID: 0000-0002-3823-0683 2022 , 'Investigating the therapeutic efficacy of non-anticoagulant fractions (Dp2 and Dp4) of enoxaparin following experimental traumatic brain injury', PhD thesis, University of Tasmania.

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Traumatic brain injury (TBI) is a debilitating neurological disorder that is fast becoming a global public health problem. It is prevalent among the active population (<45 years of age) and results from external forces encountered due to falls, explosions, bomb blasts, accidents, or sports-related collision, leading to brain dysfunction and neurodegeneration in most cases. With the increasing global incidence of TBI, it has been estimated that 69 million people worldwide will suffer from TBI yearly.
TBI triggers a cascade of events that are both primary and secondary injury mechanisms. While primary injury comprises the direct tissue damage from the impact, secondary injury occurs minutes, hours, or days after the initial impact. Neuroinflammation, a crucial component of the secondary injury mechanisms, contributes to ongoing neuronal degeneration and poor outcome following TBI. Trauma elicits neuroinflammatory responses including disruption of blood brain barrier (BBB), increased activation of microglia and astrocytes, infiltration of peripheral immune cells, and elevated levels of inflammatory cytokines/chemokines, as well as other neurotoxic molecules like reactive oxygen species. While all these responses on one hand are necessary for host defence and repair following injury, on the other hand, over time, they can become detrimental leading to further brain damage. Thus, it is now well established that neuroinflammation displays dual roles in the pathophysiology of TBI.
Despite extensive preclinical and clinical research into identifying and developing treatment for TBI, to date, there is no effective neuroprotective treatment available, as promising candidates have failed to replicate beneficial results in the clinic. With the suppression of deleterious neuroinflammatory processes linked to better outcomes in experimental TBI, drugs targeting the inflammatory cascade are being investigated as potential new therapies. Heparin and low molecular weight heparins (LMWHs), such as enoxaparin, have exhibited potential neuroprotective properties following administration in both clinical and experimental TBI, with studies showing reduced inflammation, decreased brain oedema, attenuated motor function impairment, and also ameliorated cognitive and neurological dysfunction. Although the exact mechanism underlying this remains unclear, there is evidence that the beneficial effects demonstrated by both heparin and enoxaparin are associated with their anti-inflammatory properties, which are separate from their well-known anticoagulant effects. However, despite these potential beneficial effects, treatment with heparinoids is often delayed or withheld from TBI patients in the clinic, due to an increased risk of cerebral haemorrhage. Importantly, the recently isolated and characterised di-saccharide and tetrasaccharide fragments of enoxaparin, Dp2 and Dp4, respectively, have robust anti-inflammatory effects but no anticoagulant activity. In in-vitro and ex-vivo studies, Dp2 and Dp4 inhibited the release of pro-inflammatory cytokines from human pulmonary epithelial cells and peripheral blood mononuclear cells, respectively. However, unlike enoxaparin, Dp2 and Dp4 do not have anticoagulant activity and would therefore pose no risk of bleeding to TBI patients. Therefore, in this thesis, I evaluated the effect of enoxaparin, Dp2 and Dp4 on acute neuroinflammation and long-term functional outcome following experimental TBI, with the hypothesis that Dp2 and Dp4 will significantly decrease the levels of neuroinflammation and improve recovery following TBI.
Firstly, I investigated the effect of enoxaparin and Dp4 on gliosis in the injured brain. I hypothesised that treatment would decrease the accumulation of activated astrocytes and microglia in the pericontusional cortex. To address this, 10–14-week-old mice were subjected to a moderate focal TBI induced by the controlled cortical impact injury model, while sham animals underwent surgery only. Enoxaparin, low and high doses of Dp4, or saline (vehicle) were administered either through continuous subcutaneous (SC) infusion or multiple intraperitoneal (IP) injections over 3 days post-TBI. Mice were killed at 3 days post-TBI and their brains were collected for immunohistochemistry using antibodies to detect GFAP, IBA-1 and CD68, to quantify astrocytes and microglia/macrophages in the pericontusional cortex. Our results demonstrated that irrespective of dose and mode of administration, enoxaparin and Dp4 did not attenuate astrocyte and microglial gliosis following experimental TBI.
To further explore the effect of these drugs on neuroinflammation, I measured the levels of pro- and anti-inflammatory cytokines and chemokines in the injured cortex. Additional cohorts of mice of the same experimental groups described above were killed at 6 hours post-TBI and protein levels of 23 cytokines and chemokines were quantified in the pericontusional cortex using BIO-RAD Bio-Plex Pro assay. As expected, I found that inflammatory mediators including IL-6, IL-1, MCP-1, G-CSF, KC, MIP-1α and RANTES, were increased after trauma; however, their levels were not reduced by enoxaparin or Dp4, regardless of mode of drug delivery. Interestingly, the levels of T-cell related cytokines, MIP- 1β and IL-9, were significantly reduced by IP injection, but not SC infusion, of enoxaparin. These results suggest that while some components of neuroinflammation are not affected, treatment with enoxaparin may decrease recruitment of T-cells into the contused brain. Since infiltrated T-cells can contribute to poor outcomes post-TBI, by blocking them, enoxaparin could potentially lead to neuroprotection.
Consequently, I undertook another study to investigate the long-term effects of treatment on functional outcomes after trauma. Here, I introduced Dp2 and the combination of Dp2 and Dp4 (Dp2+Dp4) as treatment options, to explore if both drugs together could have an additive effect, since earlier in vitro studies showed they suppressed the production of different pro-inflammatory cytokines in peripheral immune cells. Following CCI, mice received repeated SC injections of enoxaparin, Dp2, Dp4, Dp2+Dp4 or vehicle (saline) and behaviour was assessed on the ledged beam, hanging wire and open field at multiple timepoints across four weeks post-TBI. Our results revealed that none of the treatments ameliorated the motor function deficits that were observed for up to 7 days after trauma on the ledged beam. Also, there were no differences between the performances of the injured animals and sham animals on both the hanging wire task and the open field arena, so no effect of treatment on muscle strength or anxiety could be assessed.
Together, the results from these experimental studies were surprising, since it has been reported that heparin and enoxaparin have numerous beneficial effects after trauma. Therefore, to gain a more detailed insight into the efficacy of these drugs in the treatment of TBI, and to better understand our findings within this context, we performed a systematic review and meta-analysis on the available literature evaluating the potential neuroprotective effects of unfractionated heparin and LMWHs following experimental TBI. Given the relatively small number of publications, all studies reporting the effect of unfractionated heparin, desulfated heparin and LMWHs (enoxaparin and dalteparin) in experimental TBI were included irrespective of outcomes measured. Our results identified 11 studies published between 2000 and 2020, from which 23 outcomes were extracted. A meta-analysis was conducted on only two outcomes: brain oedema and neurologic function, because they had the highest number of comparisons and were more likely to best answer our research questions. SYRCLE’s risk of bias tool and a modified checklist from CAMARADES assessed study quality. Both tools revealed that while most of the included studies described baseline characteristics and documented group randomization, blinded outcome assessment or detection bias were not reported. Meta-analysis indicated that heparin and enoxaparin decreased brain oedema and improved neurologic function. While subgroup analysis found that there were no differences between the drug types, drug dose could have a small influence on decreased brain oedema, as overall heterogeneity was slightly reduced following subgroup analysis. However, due to insufficient data, subgroup analysis was not performed for neurologic function outcome. Furthermore, trim and fill analysis suggest that study heterogeneity, and not necessarily publication bias, accounted for funnel plot asymmetry. Overall, this review demonstrated that heparin and enoxaparin decreased brain oedema and improved neurologic function albeit with high levels of heterogeneity between studies. Individual studies also reported the beneficial effects of both drugs in other outcomes including reactive gliosis, apoptosis, and brain infarct volume. However, the findings from this review should be interpreted with caution as it was based on a very few numbers of studies whose study quality were mostly fair.
In conclusion, the studies in this thesis contribute to the body of research that is investigating the potential therapeutic effects of enoxaparin after trauma and suggest that its beneficial actions may not include attenuating glial activation. Furthermore, for the first time, the therapeutic effect of non-anticoagulant fractions of enoxaparin, Dp2 and Dp4, were evaluated in the brain, and no beneficial effects of these drugs were detected. However, based on the range of therapeutic actions attributed to heparin and enoxaparin following experimental TBI in the literature, further studies exploring different doses and pharmacodynamics of these drugs are warranted to more fully determine their potential as a pharmacotherapy for TBI.

Item Type: Thesis - PhD
Authors/Creators:Aiyede, ME
Keywords: Traumatic brain injury, Heparin, Enoxaparin, Neuroinflammation, Low molecular weight heparin, Pharmacy Neurotrauma, glial, therapeutics
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