Abstract
Objective
Thyroid hormones control a variety of processes in the central nervous system and influence its response to different stimuli, such as ischemic stroke. Post-stroke administration of 3,3′,5-triiodo-L-thyronine (T3) has been reported to substantially improve outcomes, but the optimal dosage and time window remain elusive.
Methods
Stroke was induced in mice by transient middle cerebral artery occlusion (tMCAO), and T3 was administered at different doses and time points before and after stroke.
Results
We demonstrated a dose-dependent protective effect of T3 reducing infarct volumes with an optimal T3 dosage of 25 μg/kg. In addition, we observed a time-dependent effectiveness that was most profound when T3 was administered 1 h after tMCAO (P < 0.001), with a gradual reduction in efficacy at 4.5 h (P = 0.066), and no reduction in infarct volumes when T3 was injected with an 8-h delay (P > 0.999). The protective effect of acute T3 treatment persisted for 72 h post-tMCAO (P < 0.01) and accelerated the recovery of motor function by day 3 (P < 0.05). In-depth investigations further revealed reduced cerebral edema and diminished blood–brain barrier leakage, indicated by reduced extravasation of Evans blue and diminished aquaporin-4 expression.
Conclusion
Our findings suggest that T3 may be a promising intervention for ischemic stroke in the acute phase.
Introduction
Ischemic stroke remains a major global health problem, characterized by its serious consequences and significant societal burden with 12.2 million strokes occurring every year (1). The gold standard treatment for eligible patients is intravenous thrombolysis with tissue plasminogen activator (tPA), administered within the first 4.5 h of symptom onset (2). In addition, mechanical thrombectomy within 24 h after stroke onset is indicated for patients with acute ischemic stroke due to a large artery occlusion in the anterior circulation (3). However, these treatment options are only accessible to a very limited number of patients, leading to a high demand for research into new stroke therapies.
There is increasing evidence that thyroid hormones (TH) play a significant role in the pathophysiology of ischemic stroke. Hypothyroidism has been reported to be a protective factor in ischemic stroke patients (4, 5) and rats (6), while hyperthyroidism was seen as a risk factor for poor functional outcomes in patients (7) and rats (8, 9). However, hyperthyroid rats showed profound alterations in the cardiovascular system, including hypertension and tachyarrhythmia, and TH treatment resulted in a catabolic metabolism (8), making it difficult to directly link hyperthyroidism to increased infarct volumes.
So far, only a few studies have used TH as a therapeutic treatment in experimental ischemic stroke, yet indicating a protective effect of TH when administered before (10, 11) or after ischemic stroke (11, 12, 13). In case of an ischemic insult under euthyroid conditions, acute administration of exogenous TH may avoid neuronal cell damage and reduce infarct volumes, suggesting that timing of modulation of TH action is crucial, possibly at a local level (11). However, these experiments were performed in different species (mice and rats) using different iodothyronines (T3, T4 and T2), doses (e.g., 11, 12, 25, 50 and 200 μg/kg), times of application (before or after ischemia and single injection or repeated injections) and application routes (intravenous or intraperitoneal).
Here, we performed a systematic analysis of TH effects in ischemic stroke to evaluate the optimal dose and time window for intervention. We subjected C57BL/6N mice to either 30 or 60 min of transient middle cerebral artery occlusion (tMCAO), treated them with different doses of T3 at the time of reperfusion and analyzed infarct volumes and neurological deficits 24 or 72 h post-stroke. The most effective dose (25 μg/kg) was subsequently injected at different time points either before or after stroke induction to evaluate the most effective time window. Mice treated with the most effective dose at the most effective time point (1 h after tMCAO) were further analyzed for blood–brain barrier (BBB) integrity, inflammation, thrombosis and cell death.
Methods
A detailed description of methods is given in the Supplemental materials (see section on Supplementary materials given at the end of the article).
Study design
This was a randomized, controlled study in 128 male and 20 female C57BL/6N mice (Charles River, Germany) at the age of 10–16 weeks. The number of animals was calculated via a priori sample size analysis using the G*Power 3.1 software (https://www.psychologie.hhu.de/arbeitsgruppen/allgemeine-psychologie-und-arbeitspsychologie/gpower.html). The groups were assigned randomly. Surgery, behavioral assessments and evaluation of readout parameters were performed by an observer blinded to the group allocation, with unblinding before statistical analysis. Criteria for exclusion are provided in the Supplemental material.
All animal experiments were performed in agreement with the ARRIVE (14) and the IMPROVE (15) guidelines, approved by the local state authorities (Landesamt für Natur, Umwelt und Verbraucherschutz NRW, LANUV; authorization 81-02.04.2022.A377) and conducted in agreement with the German Animal Welfare Act (German Ministry of Agriculture, Health and Economic Cooperation).
tMCAO
Cerebral ischemia was induced by either 30- or 60-min tMCAO, as described previously (16). For sham-operated mice, the same surgical procedure was conducted, excluding the insertion of the monofilament. A detailed description for surgery and calculation of infarct volume and edema is provided in the Supplemental material.
Triiodothyronine treatment
Mice received 3,3′,5-triiodo-L-thyronine (T3) (T6397; Sigma-Aldrich, Germany) as described in the Supplemental material.
Functional outcomes
Functional assessment was conducted 24 or 72 h post-tMCAO using the Bederson score (17) and a modified Neuroscore (18). Scores were assigned based on the criteria listed in the Supplemental material.
Fluorescence in situ hybridization (FISH)
To assess the T3 transition across the BBB and its activity in the brain, Krüppel-like factor 9 (Klf9) mRNA served as a marker gene for T3 action and was visualized through hybridization chain reaction (HCR) fluorescent in situ hybridization, as described elsewhere (19). Buffers, probes and hairpins were purchased from Molecular Instruments and were applied according to the manufacturer’s instructions. For quantification, the integrated Klf9 signal density was measured in two areas of the cortex. Two slices were analyzed per animal and a mean value of all signals was calculated for each hemisphere. A detailed description is given in the Supplemental materials.
Evans blue
To determine the BBB leakage, Evans blue extravasation was measured as described in the supplementary material.
Western blot
Immunoreactivity for aquaporin-4 (1:1000; ab81355; Abcam, UK) and beta-actin (1:10,000; ab8227; Abcam, UK) was detected by Western blot and quantified by densitometry. For all Western blots, beta-actin was used as a loading control.
Immunohistology
Brain sections of 20 μm thickness were fixed in 4% paraformaldehyde and then blocked for 1 h in 5% bovine serum albumin (BSA) with 0.2% Triton-X-100 in PBS, followed by overnight incubation at 4 °C with primary antibodies diluted 1:100 in PBS containing 1% BSA (anti-CD11b (MCA711, Serotec, Germany), anti-CD31 (MCA2388, Bio-Rad, Germany), anti-GPIX (M051-0, Emfret Analytics, Germany) or anti-Ly6G (127601, Biolegend, USA)). As a secondary antibody, an Alexa 488-conjugated donkey anti-rat antibody (A21208, Life Technologies, USA), diluted 1:100 in PBS containing 1% BSA, was used.
Apoptotic neurons in the ischemic hemisphere were visualized using TUNEL at 24 h after tMCAO. Brain sections were treated with a primary mouse antibody to NeuN (MAB377, Millipore, Germany, 1:1000 in PBS) and a secondary DyLight 488-conjugated goat anti-mouse antibody (ab96871, Abcam; 1:500 in PBS). TUNEL-positive cells were stained using the in situ cell death detection kit tetramethylrhodamine (TMR) red (11684795910; Sigma-Aldrich, Germany), following the manufacturer’s instructions.
Statistical analysis
GraphPad Prism software (v9.0.0; https://www.graphpad.com) was used for statistical analyses and visualization of the results. Normal (Gaussian) data distribution was tested with the D’Agostino and Pearson omnibus normality test. Two groups were compared using the unpaired, two-tailed Student’s t-test. For comparison of multiple groups, one-way analysis of variance (ANOVA) tests with post hoc Bonferroni adjusted t-tests were used for normally distributed data, and Kruskal–Wallis tests with post hoc Dunn multiple comparison tests were used for non-normally distributed data. Individual Neuroscore criteria were compared by two-way ANOVA with post hoc Bonferroni adjusted t-tests. Non-normally distributed data were presented as scatter plots, with median and normally distributed data as mean ± standard deviation. P values <0.05 were considered significant (*P < 0.05; **P < 0.01; ***P < 0.001).
Results
Evaluation of effective T3 doses for intervention 24 h after brain ischemia
Adult male C57BL/6N mice underwent a 60-min tMCAO procedure, followed by treatment with different doses of T3 (10, 25 or 50 μg/kg body weight) or 0.9% NaCl as vehicle control 1 h after occlusion (by the time of reperfusion), aiming to identify the most effective dose for acute-phase intervention. Examination of infarct volumes (2,3,5-triphenyltetrazolium chloride (TTC) staining) revealed a significant reduction in both the 25 μg/kg (mean 38.01 ± 22.64 mm3, n = 12; P < 0.001) and 50 μg/kg (mean 40.22 ± 15.88 mm3, n = 10; P < 0.01) T3-treated groups 24 h post-tMCAO compared to the controls (mean 76.93 ± 17.10 mm3, n = 10). However, the administration of 10 μg/kg T3 failed to yield a statistically significant reduction in infarct volume (mean 54.27 ± 23.31 mm3, n = 8; P = 0.081, Fig. 1A). The assessment of functional outcomes based on the Bederson score also failed to demonstrate significant improvements after T3 treatment (Fig. 1B). Since a significant protection could already be achieved with a T3 dose of 25 μg/kg and could not be further improved by applying 50 μg/kg T3, the lower dose of 25 μg/kg was used for further experiments to keep possible side effects as low as possible.
Triiodothyronine (T3) decreases infarct volume after tMCAO and improves neurological function. (A) Representative TTC stainings of coronal sections from 10-to 16-week-old male mice subjected to 60-min tMCAO, treated with either vehicle or 10, 25 or 50 μg/kg T3 1 h after tMCAO. Administration of 25 or 50 μg/kg T3 significantly reduced infarct volume (white), while a T3 dose of 10 μg/kg had no beneficial effect (n = 7–12 per group; **P < 0.01, ***P < 0.001, one-way ANOVA). (B) The assessment of neurological function by Bederson score revealed no significant improvement by T3 treatment. (C) Representative TTC stainings of coronal sections of 10- to 16-week-old male mice subjected to 60-min tMCAO and treated with either 0.9% NaCl as vehicle or 25 μg/kg T3 at different time points (1 h before or 1, 4.5 or 8 h after tMCAO). Treatment before tMCAO had no beneficial effect, while administration 1 h after tMCAO had the most significant impact on infarct volume. Administration 4.5 h later still achieved a modest reduction, but after 8 h, no beneficial effect could be observed (n = 7–12 per group; ***P < 0.001, one-way ANOVA). (D) No significant T3 effects on the neurological function, assessed by Bederson score, were observed. (E) To assess the persistence of the T3 effect, male mice were subjected to 30-min tMCAO and either T3 or vehicle was administered 1 h after occlusion. The mice were sacrificed on day 3 after tMCAO. The TTC staining demonstrates a protective effect of T3 on infarct volume for at least 3 days (n = 15–16 per group; **P < 0.01, unpaired Student’s t-test), (H) resulting in improved neuromotor function as indicated by lower Bederson scores by day 3 (n = 15–16 per group; *P < 0.05, unpaired Student’s t-test). (G) FISH with probes against Klf9 mRNA was performed on 20-μ- thick coronal brain slices to determine whether the administered T3 crosses the BBB and acts in the brain in mice treated with 25 μg/kg T3 at 1 h post-tMCAO. Klf9 levels were increased in both the ipsilateral and contralateral hemisphere after T3 administration compared to the vehicle control (n = 5 per group; *P < 0.05, **P < 0.01, one-way ANOVA).
Citation: European Thyroid Journal 14, 1; 10.1530/ETJ-24-0143
Evaluation of the most efficient time window for T3 intervention after brain ischemia
In order to determine the optimal treatment time, we administered T3 (25 μg/kg) at various time points: one hour prior, one hour after, 4.5 h after or 8 h after induction of tMCAO. Administration of T3 before the onset of brain ischemia failed to confer any protective effect on infarct size (mean: 66.83 ± 27.61 mm3, n = 10; P = 0.695). The most effective time point for intervention was at the time of reperfusion, 1 h after tMCAO, resulting in a significant reduction in infarct volume (mean 38.01 ± 22.64 mm3, n = 12; P < 0.001). Administration of T3 after 4.5 h, the maximal time window for lysis with recombinant tPA (rtPA) in humans, still resulted in a mild reduction in infarct volume (mean: 51.01 ± 8.72 mm3, n = 8; P = 0.066), albeit less pronounced. However, when administered 8 h after tMCAO, T3 treatment did not yield any alteration in infarct volume (mean: 74.11 ± 21.52 mm3, n = 8; P = 0.996, Fig. 1C). The degree of protection did not correlate with the improvement in neurological outcome, assessed by Bederson scores (Fig. 1D).
T3 improved outcome within 72 h after stroke onset
To investigate whether the administration of T3 at the time of reperfusion attenuated the damage caused by stroke or merely delayed its occurrence, the outcome after 3 days was examined. In this experiment, we used a shorter middle cerebral artery occlusion (MCAO) time of only 30 min to achieve a higher survival rate until day 3. 25 μg/kg of T3 was administered 1 h after occlusion, and the mice were monitored daily, until being sacrificed on the third day for infarct volume determination via TTC staining. In contrast to the control group, a reduction in infarct volume following a single administration of T3 (25 μg/kg) was still evident three days after tMCAO (vehicle – mean: 30.52 ± 19.74 mm3, n = 16 vs T3-treated group – mean: 10.78 ± 14.07 mm3, n = 15; **P < 0.01, Fig. 1E). T3-treated animals displayed an enhanced functional recovery compared to vehicle controls three days after tMCAO, as evidenced by a reduction in the Bederson score by day 3 (vehicle – median score: 2, n = 16 vs T3-treated group: median score 1, n = 15; P < 0.05, Fig. 1F).
To check whether T3 actually reaches the brain and modifies the expression of T3-regulated genes, we analyzed neuronal mRNA expression of Klf9 as a surrogate marker for local T3 activity in the affected (ipsilateral) and unaffected (contralateral) hemispheres by FISH in mice treated with 25 μg/kg at 1 h after tMCAO. We observed increased neuronal Klf9 transcription in T3-treated mice compared to vehicle controls both around the infarct core in the penumbra (25% increase; P < 0.05) and in the contralateral hemisphere (40% increase; P < 0.01) 24 h after tMCAO (Fig. 1G).
Validation of T3 treatment in female mice
Sex can have a significant impact on stroke outcome in rodents and in humans (20). To clarify a potential sex difference in T3 effects on stroke size, we also subjected 10- to 16-week-old female C57BL/6N mice to 60 min of tMCAO, followed by injection of 25 μg/kg T3 or 0.9% NaCl at 1 h after stroke induction. In line with the results in male mice, TTC staining in female mice revealed a notable reduction in infarct volume 24 h post-tMCAO in the T3-treatment group (mean: 42.89 ± 19.20 mm3, n = 9) compared to the control group (mean: 71.43 ± 24.41 mm3, n = 7; P < 0.05, Fig. 2A). The reduced infarct volumes, however, did not translate into a better motor function (Fig. 2B).
Triiodothyronine (T3) improves the neurological function after tMCAO. (A) 10- to 16-week-old female mice underwent 60-min tMCAO and were subsequently treated with 25 μg/kg T3 1 h after tMCAO. The TTC staining revealed significantly reduced infarct volumes (n = 7–9 per group; *P < 0.05, unpaired Student’s t-test) (B) but no significant effects on neurological function on day 1. (C) The combination of data obtained from male and female mice confirmed a sex-independent effect of T3 on infarct volume 24 h after tMCAO (n = 17–21 per group; ***P < 0.001; unpaired Student’s t-test). Data of male mice are presented in black, while data of females are shown in white. (D) The combined data also confirmed a significant improvement in neurological outcome by T3 on day 1 after tMCAO (n = 17–21 per group; *P < 0.05; unpaired Student’s t-test). (E) T3-treated male mice displayed an improved neurological function assessed by the modified Neuroscore compared to controls 24 h after 60 min of tMCAO. Sham-operated animals received scores of zero in all five criteria (n = 5–7 per group; *P < 0.05, ***P < 0.001, one-way ANOVA). (F) Individual depictions of the five evaluated criteria for the modified Neuroscore (spontaneous activity, body symmetry, gait, circling and fibrillary response) for T3-treated and control mice reveal a functional improvement in terms of body symmetry, gait and circling 24 h after tMCAO (n = 5–7 per group; *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA).
Citation: European Thyroid Journal 14, 1; 10.1530/ETJ-24-0143
However, by combining the data from male and female mice, in addition to the reduction in infarct volume by T3 (Fig. 2C), we were able to show significant improvement in neurological outcome according to the Bederson score (median score 3 for vehicle-treated (n = 17) vs 2 for T3-treated (n = 21); P < 0.05; Fig. 2D) that both were independent of sex. Subsequently, an additional group of male mice was treated with T3 vs vehicle and subjected to the modified Neuroscore evaluation, which allows differentiation of subtle differences in neurological deficits. This revealed significant differences in functional improvement (median score 10 for vehicle-treated (n = 5) vs 7 for T3-treated (n = 7); P < 0.05) at 24 h post-tMCAO (Fig. 2E), especially in terms of body symmetry (P < 0.05), gait (P < 0.001) and circling (P < 0.01; Fig. 2F).
T3 administration reduced BBB leakage
Next, we determined whether T3 affects BBB integrity. After ischemia, the BBB opens within hours, permitting the influx of large and small molecules into the brain (21). The injection of the azo dye Evans blue allows to assess the degree of BBB breakdown as a consequence. Significantly less Evans blue was visible in the brain tissue of mice treated with 25 μg/kg 1 h post-tMCAO (mean: 229.6 ± 42.9 ng/mg, n = 10) compared to controls (mean: 306.2 ± 79.1 ng/mg, n = 7; P < 0.05, Fig. 3A), with no differences contralaterally, suggesting amelioration of BBB damage in the acute phase.
Triiodothyronine (T3) reduced BBB leakage and diminished edema. (A) 12-week-old male mice underwent tMCAO and were treated with T3 or vehicle 1 h after tMCAO. Evans blue dye was intravenously injected 22 h after tMCAO to evaluate the integrity of the BBB and animals were killed two hours later. Mice treated with T3 exhibited significantly reduced Evans blue extravasation compared to vehicle controls, while no differences were observed contralaterally (n = 7–10 per group; *P < 0.05, unpaired Student’s t-test). (B) Edema was assessed from coronal brain sections. Edema formation on day 1 after tMCAO was significantly diminished following T3 administration compared to controls (n = 7–9; **P < 0.01, unpaired Student’s t-test). (C) On day 3 after tMCAO, only a modest decrease in edema formation with no statistical significance could be observed after T3 treatment with occlusion time reduced to 30 min (n = 15–16 per group; P = 0.063, unpaired Student’s t-test). (D and E) Representative aquaporin-4 (Aqp4) Western blot bands of control (C), T3-treated (T3) or sham-operated (S) mice 24 h after tMCAO. The marker (M) displays a fluorescent band at 70 kDa in basal ganglia and cortex on the actin western blots. (F) Aquaporin-4 expression increased 1.5-fold after tMCAO in the basal ganglia in vehicle-treated mice compared to sham-operated mice. Treatment with 25 μg/kg T3 reduced aquaporin-4 expression below the sham level (n = 3–5 per group; **P < 0.01, one-way ANOVA). (G) Aquaporin-4 expression in the cortex tripled in the vehicle controls compared to the sham-operated mice. T3 administration reduced this expression below the sham levels (n = 3–5 per group; *P < 0.05, ***P < 0.001, one-way ANOVA).
Citation: European Thyroid Journal 14, 1; 10.1530/ETJ-24-0143
In addition, the T3-treated mice exhibited significantly less edema after 24 h following 60-min tMCAO (mean: 1.94 ± 1.84%, n = 9) in contrast to controls (mean: 6.46 ± 3.01%, n = 7; P < 0.01, Fig. 3B). However, on day 3 following 30-min tMCAO, the T3-treated mice displayed no edema reduction (mean: 3.94 ± 1.887%, n = 15) compared to controls (mean: 6.270 ± 4.30%, n = 16; P = 0.063, Fig. 3C).
To elucidate the potential underlying mechanisms of BBB stabilization and edema formation, aquaporin-4 expression following tMCAO was assessed through Western blot analysis. Aquaporin-4 expression increased 1.5-fold in the basal ganglia and up to 3-fold in the cortex after tMCAO in control mice compared to sham-operated animals. Conversely, T3 treatment significantly reduced aquaporin-4 expression below sham levels, both in the cortex and basal ganglia in the ipsilateral hemisphere 24 h after tMCAO (Fig. 3D, E, F, G). This suggests a protective effect of T3 treatment in brain ischemia by ameliorating edema formation.
T3 administration reduced inflammation, thrombosis and cell death
To assess the potential impact of T3 treatment on local inflammation and the infiltration of immune cells, immunostainings were conducted 24 h following stroke onset to visualize the Ly6G-positive neutrophil granulocytes and CD11b-positive macrophages/microglia. Following T3 administration, a significant reduction in the infiltrated Ly6G-positive neutrophil granulocytes in the ischemic hemispheres was noted (mean: 111 ± 29.3 in controls (n = 5) vs 34 ± 28.0 in the T3-treated group (n = 6); P < 0.05, Fig. 4A). However, no discernible differences were observed in the number of CD11b-positive cells (mean: 140 ± 51.3 in vehicle controls (n = 5) vs 113 ± 78.0 in the T3-treated group (n = 6); P = 0.521; Fig. 4B).
Triiodothyronine (T3) decreases inflammation and apoptosis. Representative pictures of fluorescence immunostainings that were conducted to evaluate the inflammatory responses and the fraction of occluded microvessels on day 1 after tMCAO. (A) The number of Ly6G+ neutrophils within the ipsilateral hemisphere was significantly reduced in the T3-treated animals compared to controls, (B) while staining for Cd11b+ macrophages revealed no significant differences (n = 5–6 per group; *P < 0.05, unpaired Student’s t-test). (C) To address apoptotic neurons, a cell death assay was conducted together with NeuN immunofluorescence staining. The TUNEL+ area (in red) was quantified and compared between groups. T3-treated mice displayed significantly less apoptosis compared to vehicle controls (n = 6–7 per group; *P < 0.05, unpaired Student’s t-test). (D) The staining for vessels (CD31) and platelets (GPIX) revealed that after T3 administration, the proportion of microvessels occluded by thrombi was lower than in vehicle controls. This assessment was conducted in four representative areas within the penumbra (n = 6–7 per group; *P < 0.05, unpaired Student’s t-test).
Citation: European Thyroid Journal 14, 1; 10.1530/ETJ-24-0143
Next, apoptotic neurons were examined via NeuN and TUNEL costaining to investigate the impact of T3 on DNA fragmentation as a consequence of cell death. The area fraction occupied by apoptotic neurons per slice diminished to one-third after T3 administration (mean % TUNEL+ area: 21.97 ± 3.48% in vehicle controls (n = 6) vs 7.70 ± 3.36% after T3 administration (n = 7); P < 0.05, Fig. 4C).
Furthermore, we examined whether reduced intracerebral thrombus formation was also one of the underlying mechanisms. In histological brain sections stained for the endothelial marker CD31 and the platelet marker GPIX, about 20% (mean: 22.64 ± 5.14%) of all vessels in the ischemic hemisphere were occluded by microthrombi in control mice. In contrast, T3-treated mice showed significantly increased microvascular patency, with less occluded vessels (mean: 6.35 ± 3.7% after T3 treatment (n = 7) vs 22.64 ± 5.14% in vehicle controls (n = 6); P < 0.05, Fig. 4D).
Discussion
Our study focused on the potential benefits of T3 administration in a murine model of tMCAO. Treatment with T3 after reperfusion resulted in smaller brain infarcts and enhanced neurological outcomes. We also showed that this effect was independent of sex (Fig. 2C). Prior research has suggested that administration of TH can improve acute-phase outcomes and decrease infarct volumes after tMCAO (10, 11, 12). In our study, we evaluated the dose- and time-dependent effects of T3 application after brain ischemia, which to date has not been fully addressed. While T3 administration 1 h after tMCAO at a dosage of 10 μg/kg did not yield a significant reduction in infarct volume, both 25 and 50 μg/kg showed substantial improvements (Fig. 1A). The 25 μg/kg dose aligns with that used by Sadana et al., who induced 60-min tMCAO in male CD1 mice and injected 25 μg/kg T3 intravenously 10–15 min after reperfusion (11). Application of T3 within 4.5 h of tMCAO, which corresponds to the clinical rtPA time window for intervention in humans, showed a nonsignificant trend to reduction (P = 0.066) but had no beneficial effects when given at a later time point (Fig. 1C).
In contrast to other studies (10, 11), we did not observe any protective effect of T3 when administered before stroke onset (Fig. 1C). This could be due to the fact that the time of pretreatment was 1 h before induction of stroke in our study, but only 30 min in other studies. The protective, noncanonical effect is presumably mediated via the PI3K/Akt signaling pathway and eNOS, an important downstream target of Akt, both responding rapidly (within the first 60 min) to T3 stimulation (10, 29). Another reason could be the use of different mouse strains (C57BL/6N mice in our study and in that by Hiroi et al. vs CD1 mice used by Sadana et al.) or different times of MCA occlusion (1 h in our study and in that by Sadana et al. vs 2 h in the study by Hiroi et al.) (10, 11). On the other hand, T3 administration 1 h before tMCAO did not lead to an increase in infarct volume compared to the control animals. Since hyperthyroid rats develop larger infarcts than euthyroid rats (8), one could envisage that pretreatment with T3 also leads to larger infarcts. However, it appears that a single administration of T3 before stroke onset is not sufficient to worsen stroke outcome.
Importantly the benefits of T3 application 1 h after tMCAO persisted for the first three days with reduced occlusion time, showing that T3 treatment did not delay but protected from infarct growth. In addition to the reduction in infarct volume, the treated mice also displayed accelerated recovery of motor function by that time (Fig. 1E). Whether these protective effects of a single T3 injection can persist over a longer time period remains to be elucidated. So far, there are no studies addressing stroke outcome beyond 24 h, when T3 was administered at the time of reperfusion. There are two studies assessing later times after stroke in the context of TH (23, 24). These studies focused on regenerative processes, such as neuronal regeneration, and T3 was administered after the infarct had matured. Consequently, there is no statement on infarct development. A single T3 administration 24 h after tMCAO in rats increased the expression of brain-derived neurotrophic factor, Nestin and SOX2 on day 7 in the subventricular zone (22), possibly enhancing recovery after an ischemic stroke. Talhada and colleagues tested the effects of long-term T3 administration after photothrombosis (23). Intraperitoneal injections of T3 at a dose of 50 μg/kg were started at day 2 post-stroke and were repeated every second day until the end of the experiment on day 14. This treatment significantly improved sensorimotor function in the rotating pole test, without affecting the infarct size. Improved motor function was explained by increased dendritic spine density and modulated synaptic neurotransmission by increased levels of synaptotagmin 1 and 2 and the GluR2 subunit of AMPA receptors and increased dendritic spine density. Enhancement of neurotrophic factors was also seen 24 h after stroke in rats treated with a single dose of thyroxine (13). Taken together, these studies suggest an influence of TH on neuronal regeneration after stroke and highlight that further pharmacokinetic studies are warranted to fully explore when, how long or in which interval T3 may exert beneficial effects on ischemic brain injury.
One of the major complications after brain ischemia is the breakdown of the BBB, leading to inflammatory processes, leukocyte infiltration and the formation of brain edema. Consequently, secondary infarct growth and further deterioration of neurological symptoms occur (24, 25). Interestingly, BBB integrity was well maintained after T3 treatment, as indicated by reduced Evans blue extravasation and less edema that persisted over three days (Fig. 3A, B, C). The water channel protein aquaporin-4 is critically involved in the formation of brain edema after ischemic stroke (26). We identified a reduction in aquaporin-4 expression following T3 treatment in the acute phase of ischemia as one possible reason for ameliorated edema formation (Fig. 3D, E, F, G). This is in line with a previous study, where T3 treatment 10–15 min after reperfusion decreased edema formation via a negative regulatory influence on aquaporin-4 expression in CD1 male mice after stroke (11). Negative regulation of aquaporin-4 was also seen in glioblastoma cell lines after stimulation with 50 nM T3 (27).
Another consequence of a disrupted BBB is an increase in inflammatory processes and leukocyte infiltration. A reduced expression of proinflammatory tumor necrosis factor alpha and interleukin-6 has been observed on day 4 after stroke in rats treated with T3 (21). Neutrophils are among the first cells to invade parenchyma and play a causative role in infarct development. Indeed, we observed reduced neutrophil infiltration into the brain parenchyma after T3 treatment, indicating a reduced inflammatory response (Fig. 4A). Moreover, neutrophils can interact with platelets and endothelial cells and impair tissue reperfusion, a phenomenon commonly referred to as ‘no reflow phenomenon’ (28). In our study, a higher microvascular patency with fewer thrombi in the microvasculature was observed in mice treated with T3 (Fig. 4D). An alternative explanation could be a direct effect of T3 on endothelial cells via noncanonical T3 action, as was previously shown by Geist et al. (29).
Immune cells can also secrete cytokines, stimulate neuronal cell death and promote tissue damage after brain ischemia (30). Administration of thyroxine can have anti-apoptotic effects after ischemic stroke in rats by decreasing the expression of pro-apoptotic Bax and increasing anti-apoptotic B-cell lymphoma 2 (13). An anti-apoptotic effect of T3 was also confirmed in our study, as seen by a reduced number of DNA-fragmented cells in the ischemic brain after T3 treatment (Fig. 4C).
In summary, in our study, we found that a single dose of T3 administered post-tMCAO given within 1 h after the event reduced infarct volume and improved neurological outcome, with a lasting effect over at least 3 days. The rapid onset of beneficial effects strongly suggests noncanonical T3 action. Possible underlying mechanisms of the protective effect include stabilization of the BBB, along with reduced aquaporin-4 expression, a reduced inflammatory response and less cell death.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/ETJ-24-0143.
Declaration of interest
The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Funding
This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) in the framework of SFB/TR 296 LOCOTACT – Project-IDs 424957847 (FL, CK, DF, HH and SM) and FOR 2879 ImmunoStroke - Project number: 405358801 (CK and FL).
Author contribution statement
FL, DF and CK conceived this study. DU, FL, LIS, ML, SH and RS performed the experiments and analyzed the data. DU, FL and DF wrote the manuscript. HH and SM provided reagents. TH, HH, MS and SM revised the manuscript.
Acknowledgements
The authors thank the Imaging Core Facility Essen (IMCES) for support with the Olympus microscope. We are also grateful to Stefanie Hezel and Kristina Wagner for their dedicated technical support.
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