Preclinical Evaluation of Fingolimod in Rodent Models of Stroke With Age or Atherosclerosis as Comorbidities.

Preclinical data indicate that fingolimod improves outcome post-ischaemia. This study used a rigorous study design in normal male C57BL/6JOlaHsd mice and in mice with common stroke comorbidities to further evaluate the translational potential of fingolimod. Stroke was induced via middle cerebral artery electrocoagulation in 8-9-week old mice (young mice), 18 month old mice (aged mice), and in high-fat diet-fed 22-week old ApoE-/- mice (hyperlipidaemic mice). Recovery was evaluated using motor behavioural tests 3 and 7 days after stroke. Tissue damage was evaluated at 7 days. A lower dose of fingolimod, 0.5 mg/kg, but not 1 mg/kg, increased lesion size but decreased ipsilateral brain atrophy in younger mice, without an effect on behavioural outcomes. Fingolimod-treated aged mice showed a significant improvement over saline-treated mice in the foot fault test at 7 days. Fingolimod-treated hyperlipidaemic mice showed a decreased infarct size but no difference in behavioural performance. Increasing fingolimod treatment time to 10 days showed no benefit in young mice. Pooled data showed that fingolimod improved performance in the foot fault test. Flow cytometry studies showed that fingolimod had marked effects on T cell frequencies in various tissues. The results show that the effects of fingolimod in stroke are less robust than the existing literature might indicate and may depend on the inflammatory status of the animals.

Histological, behavioural and flow cytometric datasets relating to acute ischaemic stroke in young, aged and ApoE-/- mice in the presence and absence of immunomodulation with fingolimod.

In this work, the sphingosine-1-phosphate receptor modulator fingolimod was assessed as a preclinical candidate for the treatment of acute ischaemic stroke according to the Stroke Therapy Academic Industry Roundtable (STAIR) preclinical recommendations. Young (15-17 weeks), aged (72-73 weeks), and ApoE-/- mice (20-21 weeks) fed a high fat diet (all C57BL/6 mice) underwent permanent electrocoagulation of the left middle cerebral artery. Mice received either saline or fingolimod (0.5 mg/kg or 1 mg/kg) at 2-, 24-, and 48-hours post-ischaemia via intraperitoneal (i.p.) injection. Another cohort of young mice (8-9 and 17-19 weeks) received short-term (5 days) or long-term (10 days) fingolimod (0.5 mg/kg) treatment in a treatment duration study. For young, aged, and ApoE-/- mice, motor behavioural tests (cylinder and grid-walking) were performed at days 0, 3, and 7 post-ischaemia to evaluate neurobehavioural recovery. In the treatment duration study, the grid-walking test was performed at days 0, 2, 5 and 10 post-ischaemia. Brain tissue sections were stained with haematoxylin and eosin (H&E), and NeuN to quantify tissue damage. Flow cytometry was used to quantify T cell populations in blood, spleen, and lymph nodes. The data presented in this article improves our understanding of the potential neuroprotective and immunomodulatory effects of fingolimod in a mouse model of brain ischaemia. Such data may be significant in the design of future preclinical and clinical stroke studies for fingolimod.

The effect of fingolimod on regulatory T cells in a mouse model of brain ischaemia.

BACKGROUND: The role of the immune system in stroke is well-recognised. Fingolimod, an immunomodulatory agent licensed for the management of relapsing-remitting multiple sclerosis, has been shown to provide benefit in rodent models of stroke. Its mechanism of action, however, remains unclear. We hypothesised fingolimod increases the number and/or function of regulatory T cells (Treg), a lymphocyte population which promotes stroke recovery. The primary aim of this study was to rigorously investigate the effect of fingolimod on Tregs in a mouse model of brain ischaemia. The effect of fingolimod in mice with common stroke-related comorbidities (ageing and hypercholesteremia) was also investigated. METHODS: Young (15-17 weeks), aged C57BL/6 mice (72-73 weeks), and ApoE-/- mice fed a high-fat diet (20-21 weeks) underwent permanent electrocoagulation of the left middle cerebral artery. Mice received either saline or fingolimod (0.5 mg/kg or 1 mg/kg) at 2, 24, and 48 h post-ischaemia via intraperitoneal injection. Another cohort of young mice (8-9, 17-19 weeks) received short-term (5 days) or long-term (10 days) fingolimod (0.5 mg/kg) treatment. Flow cytometry was used to quantify Tregs in blood, spleen, and lymph nodes. Immunohistochemistry was used to quantify FoxP3+ cell infiltration into the ischaemic brain. RESULTS: Fingolimod significantly increased the frequency of Tregs within the CD4+ T cell population in blood and spleen post-ischaemia in all three mouse cohorts compared to untreated ischemic mice. The highest splenic Treg frequency in fingolimod-treated mice was observed in ApoE-/- mice (9.32 ± 1.73% vs. 7.8 ± 3.01% in young, 6.09 ± 1.64% in aged mice). The highest circulating Treg frequency was also noted in ApoE-/- mice (8.39 ± 3.26% vs. 5.43 ± 2.74% in young, 4.56 ± 1.60% in aged mice). Fingolimod significantly increased the number of FoxP3+ cells in the infarct core of all mice. The most pronounced effects were seen when mice were treated for 10 days post-ischaemia. CONCLUSIONS: Fingolimod increases Treg frequency in spleen and blood post-ischaemia and enhances the number of FoxP3+ cells in the ischaemic brain. The effect of fingolimod on this regulatory cell population may underlie its neuroprotective activity and could be exploited as part of future stroke therapy.

Acute Treatment With Fingolimod Does Not Confer Long-Term Benefit in a Mouse Model of Intracerebral Haemorrhage.

Intracerebral haemorrhage (ICH) has no specific treatment, but accounts for up to 15% of all strokes and has the highest mortality. Fingolimod (FTY720) is an immunomodulator approved for the management of multiple sclerosis, with abundant evidence of efficacy in experimental ischemic stroke, and more limited evidence in experimental ICH. The goal of this study was to confirm the efficacy of fingolimod in experimental ICH using rigorous and statistically well-powered studies. ICH was induced in C57BL/6JOlaHsd male and female mice by intrastriatal bacterial collagenase injection. Fingolimod (0.5 mg/kg) or saline was administered intraperitoneally after 0.5, 24 and 72 h, in a randomized and blinded manner. Functional improvement with cylinder, wire hanging, and foot fault tests was evaluated one and two weeks later. Lesion volume and hemispheric atrophy were quantified at the 14-day endpoint. There was a higher mortality in saline-treated females compared to fingolimod-treated females and saline-treated males. There was no treatment- or gender-related difference in the behavioural tests. Histological outcome measures did not differ between any of the groups. These results, contrasting with those of previous studies of fingolimod in experimental ICH, emphasize the importance of rigorous testing of this agent in models more representative of the clinical situation.

Enhanced cardiac function in Gravin mutant mice involves alterations in the ß-adrenergic receptor signaling cascade.

Gravin, an A-kinase anchoring protein, targets protein kinase A (PKA), protein kinase C (PKC), calcineurin and other signaling molecules to the beta2-adrenergic receptor (ß2-AR). Gravin mediates desensitization/resensitization of the receptor by facilitating its phosphorylation by PKA and PKC. The role of gravin in ß-AR mediated regulation of cardiac function is unclear. The purpose of this study was to determine the effect of acute ß-AR stimulation on cardiac contractility in mice lacking functional gravin. Using echocardiographic analysis, we observed that contractility parameters such as left ventricular fractional shortening and ejection fraction were increased in gravin mutant (gravin-t/t) animals lacking functional protein compared to wild-type (WT) animals both at baseline and following acute isoproterenol (ISO) administration. In isolated gravin-t/t cardiomyocytes, we observed increased cell shortening fraction and decreased intracellular Ca(2+) in response to 1 µmol/L ISO stimulation. These physiological responses occurred in the presence of decreased ß2-AR phosphorylation in gravin-t/t hearts, where PKA-dependent ß2-AR phosphorylation has been shown to lead to receptor desensitization. cAMP production, PKA activity and phosphorylation of phospholamban and troponin I was comparable in WT and gravin-t/t hearts both with and without ISO stimulation. However, cardiac myosin binding protein C (cMyBPC) phosphorylation site at position 273 was significantly increased in gravin-t/t versus WT hearts, in the absence of ISO. Additionally, the cardioprotective heat shock protein 20 (Hsp20) was significantly more phosphorylated in gravin-t/t versus WT hearts, in response to ISO. Our results suggest that disruption of gravin's scaffold mediated signaling is able to increase baseline cardiac function as well as to augment contractility in response to acute ß-AR stimulation by decreasing ß2-AR phosphorylation and thus attenuating receptor desensitization and perhaps by altering PKA localization to increase the phosphorylation of cMyBPC and the nonclassical PKA substrate Hsp20.