Appropriate supplementation of testosterone alleviates post-stroke damage via decreasing inflammation and oxidative stress in aged male C57BL/6 mice

Abstract

Stroke injury is closely related to testosterone levels. Testosterone supplementation in elderly men is seen to protect the cardiovascular system and reduce the risk of stroke. But this medication method is controversial. This study aims to investigate the effect of long-term testosterone supplementation on brain injury after stroke in aged mice. 60 male C57BL/6 mice,12-months of age were divided into 3 groups: low-dose group, high-dose group, and control group, each group was injected subcutaneously with 100 μL of sesame oil or 5 mg/kg or 50 mg/kg of testosterone (in 100 μL of sesame oil) twice per week, respectively. One week after the injection, stroke was induced by light. After the stroke, the injection continued for 6 weeks. The motion ability was measured by rotating rod and tail suspension. The brain injury was observed by naked eyes and TTC staining. In addition, we measured the inflammation (Tnf-α, Il-6, and Mcp-1) and oxidative stress (Malondialdehyde (MDA) and T-AOC) in the injured tissue 72 h post-stroke. Low-dose testosterone supplementation improved the motion ability and decreased brain injury. It also decreased the inflammatory factors (Tnf-α, Il-6, and Mcp-1), decreased MDA product, and increased T-AOC. High-dose testosterone supplementation not only reduced the motion ability and aggravated stroke injury, but also increased the inflammation, MDA level and decreased T-AOC level. In summary, supplementation of testosterone at normal levels in elderly mice can alleviate post-stroke injury by reducing inflammation and oxidative stress; however, excessive supplementation may cause unexpected injuries. This study has important implications for the application of testosterone replacement therapy.

Introduction

Stroke is one of the leading cause of death and severe disability worldwide, ¹ and most survivors will suffer long-term functional defects.^2,3 Stroke is a sudden neurological disease, and there are few effective treatments in the emergency period. Ischemic stroke accounts for the majority of cerebrovascular events, and revascularization therapy, such as venous thrombolysis and intravascular thrombectomy is currently the main available treatment method. ⁴ Studies have shown that treating the acute phase of ischemic stroke, patients with mild to moderate dysfunction accounted for 44% and 35%, respectively, at 3 months and 12 months after recovery. ⁵ These functional deficiencies include cognitive, speech, visual, sensory, and motor deficits, of which dyskinesia is the most common symptom. ⁶ In addition, motion ability recovery after stroke is a complex, dynamic, and multi-factorial process, in which the interaction between genetic, pathophysiological, sociodemographic, and therapeutic factors determines the recovery trajectory.

Older age is generally considered to be an important prognostic factor for poor prognosis after an ischemic and hemorrhagic stroke, and nearly half of the elderly survivors perform mild to severe disability.^7,8 Compared to men, women seem to be more likely to become disabled after a stroke. ⁹ However, compared to young men, young women have better post-stroke outcomes. ¹⁰ The fundamental reason behind this gender difference is not fully understood. ¹¹

Testosterone, a steroid hormone mainly secreted by the testes, ovaries, and adrenal glands is of great significance for maintaining health.^12,13 It plays a critical role in maintaining muscle strength and quality, bone density and strength, refreshing, and improving physical fitness.^14,15 Higher testosterone levels in men affect stress response and cardiovascular diseases. Under normal circumstances, testosterone will increase the stress response, which may be an important mechanism for hormones to promote adaptive response behavior. ¹⁶ Importantly, testosterone can affect the occurrence of human cardiovascular diseases by regulating lipid metabolism.^17–21 Clinical studies have shown that endogenous androgen testosterone has an anti-atherosclerotic effect in men, while it has an atherosclerotic effect in women. ²⁰ Atherosclerosis is a high-risk factor for stroke. Similarly, exogenous testosterone also plays a protective role in male animals' cerebrovascular injury, while it has a harmful effect in female animals. ²¹ As age increases, the level of testosterone in various male mammalian species will gradually decrease.^22,23 Patients with testosterone deficiency are often companied with hyperthyroidism, liver cirrhosis, renal failure, severe trauma, and cerebrovascular diseases. Testosterone replacement therapy has become a common treatment method for patients with testosterone deficiency, with a safety record and great efficacy.^12,24,25 However, other studies have shown that the abuse of testosterone replacement therapy may increase the incidence of cardiovascular events.^12,25 At present, there is no unified conclusion.

Inflammation following tissue injury is usually divided into three stages. The first is within 48 h after injury, the injured tissue releases chemokines and cytokines to recruit monocytes. The second stage is within 1-7 days after injury, where macrophages polarize to the M1 (pro-inflammatory) subtype to clear foreign pathogens and necrotic cells. The third stage is after 5 days post-injury, when macrophages polarize to the M2 (anti-inflammatory) subtype to promote tissue repair. In addition, oxidative stress often accompanies inflammation. Overproduction of inflammation and oxidative stress may exacerbate tissue injury.^26,27 Conversely, inhibiting inflammation and oxidative stress inhibits tissue repair. Previous studies have shown that testosterone regulates vascular function by mediating inflammatory cells and oxidative stress.^28,29 It is reasonable to speculate that the exacerbation of stroke caused by abnormal testosterone levels may be related to inflammation and oxidative stress in the early stages of tissue injury.

In this study, we explored the effects of testosterone supplementation on brain injury after stroke. We hypothesized that adequate supplementation of testosterone can alleviate post-stroke injury via regulating inflammation and oxidative stress in old mice. The study was divided into three groups (control group; low-dose supplementation group; and high-dose supplementation group) to observe the effects of normal supplementation and excessive supplementation on stroke. The effects of testosterone on brain injury after stroke were evaluated by testing the motion ability, infarct size, inflammation, and oxidative stress.

Materials and methods

Ethics

All procedures and protocols were approved by the Animal Ethics Committee at Shenmu City Hospital (Ethics No. 20210424) according to the Regulations for the Care and Use of Laboratory Animals published by the Chinese Government.

Animals

Sixty 8-week-old male C57BL/6 mice were purchased from Charles River (Beijing, China). All mice were housed in groups of 4-6 animals per cage, maintained in half/half daylight/dark cycle and 25 ± 1°C room temperature, with free access to food and water. Experiment starts when the mice were 12-month-old. Mice were divided into 3 groups, the control group, low-dose group, and high-dose group. The control group received injections of 100 μL sesame oil. Low-dose group and high-dose group were administered by subcutaneous injections of 5 mg/kg or 50 mg/kg testosterone in 100 μL sesame oil; twice per week (testosterone, T102169, Aladdin, Shanghai, China). The dose of the drug was based on previous studies.^28,30,31 One week after the injection, photothrombosis was induced by illumination. Briefly, mice were anaesthetized (R510-22–10, RWD, Shenzhen, China) and fixed on a stereotaxic apparatus. The scalp was scrapped with a cut in the skull for injection and waited for 5 min after intraperitoneal injection of Rose Bengal (1 mg/mouse). Refer to previous studies for details.^32,33 After the stroke, the injection continued for 6 weeks.

Serum testosterone levels

Fresh blood was kept in a preservation tube containing sodium heparin. The supernatant was collected by centrifugation at 3000 r/min for 30 min. The mixing step was completed according to the ELISA kit instructions (QS43305, Qisong, Beijing, China) OD values were read at 450 nm.

Motion ability

Rotarod test provides a convenient way to detect the locomotor function of rodents. ³⁴ Mice recovered for 2 weeks after the surgery, went through 3 weeks of training. The mice were placed on an accelerated rotating rod (4–40 r/min; YLS-4C, Yiyan, Jinan, China), and the latency of falling from the rod was recorded.

The tight rope test is a classic method that can quickly evaluate motion ability. ³⁵ Mice were placed in the middle of the rope (60-cm), and the time it takes to reach the end of the rope or to maintain balance was recorded. The maximum testing time was kept at 60 s.

Infarct volume

Mice were perfused with sterile saline. Brains were preserved at-20°C for 20 min, then placed in a fixed container and cut into 2 mm pieces. Samples were stained using a 2,3,5-Triphenyltetrazolium chloride (TTC) solution (G1017, Servicebio, Wuhan, China). Pictures were taken under a cellphone camera. Infarct size was quantified with software ImageJ.

Inflammation

Fresh tissue from the injury site was preserved in RNA-wait (BL621 A, Biosharp, Hefei, China) overnight and then transferred to - 80°C for long-term storage. About 25 mg tissue was taken for RNA isolation using Trizol reagent (15,596,026, Thermo, Waltham MA, USA). Complementary DNA (cDNA) was synthesized using kit (K1622, Thermo, Waltham MA, USA). Quantitative Real-time PCR (qRT-PCR) was conducted on the ABI-7500 system (Applied Biosystems) using SYBR Green qPCR master mix (B21203, Bimake, Shanghai, China) to measure mRNA expression levels, which were normalized to the expression level of Gapdh. Details were described in references. ³⁶ Primers: Tnf-α, forward 5^′- ACGTCGTAGCAAACCACCAA- 3^′, reverse 5^′- GCAGCCTTGTCCCTTGAAGA- 3^′; Il-6, forward 5^′- TCTATACCACTTCACAAGTCGGA- 3^′, reverse 5^′- GAATTGCCATTGCACAACTCTTTC- 3^′; Mcp-1, forward 5^′- CCAGCCTACTCATTGGGATCA- 3^′, reverse 5^′- CTTCTGGGCCTGCTGTTCA- 3^′; Gapdh, forward 5^′- AGGTCGGTGTGAACGGATTTG- 3^′, reverse 5^′- GGGGTCGTTGATGGCAACA- 3^′.

Oxidative stress

25 mg fresh tissue from stroke site was mixed with 250 μL saline and ruptured in an ice-water bath. The homogenate was centrifuged at 12,000 r/min for 5 min at 4°C, and the supernatant was taken for measurement. Protein concentration was measured using BCA kit (G2026, Servicebio, Wuhan, China). Malondialdehyde (MDA) was measured using the MDA kit (A003-1, Jiancheng, Nanjing, China). OD value was measured at 532 nm with a microplate reader. The total antioxidant capacity (T-AOC) was measured using the T-AOC kit (A015-2–1, Jiancheng, Nanjing, China). OD value was measure at 405 nm with a microplate reader.

Statistical analysis

Data are shown as mean ± standard deviation (SD) values. Unpaired Students’ T-test was used to test statistical significance between two groups. For experiments that include more than two groups, comparison was conducted using one-way ANOVA followed by Tukey’s test. All statistical tests were performed using GraphPad Prism (Version 6.0, Inc. Chicago, IL, USA). The p-value less than 0.05 to indicate statistical significance.*p < .05; **p < .01; ***p < .001; ****p < .0001.

Results and discussion

Aging decreases testosterone levels

To test whether ageing affects testosterone levels. We compared serum testosterone levels and testicular weights in 8-week-old (young) and 12-month-old (old) male mice. Ageing did not affect testicular weight, but significantly reduced serum testosterone levels (Figure 1(a) and (b)), suggesting that testosterone synthesis levels decrease with age.

Figure 1.

Testicular weight and testosterone levels. (a) Testis weight (young 0.1789 ± 0.005,238 vs Old 0.179 ± 0.005,944, g, n = 10). (b) Serum testosterone levels (young 5.157 ± 1.668 vs Old 3.034 ± 1.72, g, n = 10).

Testosterone supplementation elevates testosterone levels in old mice

To test whether testosterone supplementation can elevate testosterone levels in aged mice, we measured serum testosterone levels 48 h after injection. The results showed that the low-dose group still maintained normal physiological levels of testosterone. However, the physiological levels of testosterone in the high-dose group were significantly higher than the normal range (Figure 2).

Figure 2.

Physiological levels of testosterone after testosterone injection in aged mice. Control 3.055 ± 1.446 vs low 4.908 ± 0.7801 vs high 12.05 ± 2.776, ng/ml, n = 10.

Motion ability

The motion ability after stroke was measured through the rotarod test and tight rope test. Compared with the control group, the low-dose testosterone (normal supplementation) significantly enhanced the motion ability and coordination ability of the mice (Figure 3(a) and (b)). However, high-dose testosterone (excessive supplementation) did not improve the exercise ability of mice after stroke, but increased the time of the tight rope test.

Figure 3.

Low-dose testosterone supplementation improved the motion ability after stroke, and high-dose testosterone supplementation reduced the motion ability. (a) Rotarod test (control 76 ± 13.39 vs low 98.2 ± 13.26 vs high 76.4 ± 12.45, seconds, n = 10; CL p = 0.0399, LHP = 0.0391, CHP = 0.9456). (b) Tight rope test (control 4.923 ± 1.493 vs Low 5.304 ± 1.438 vs high 3.762 ± 0.845, seconds, n = 10; CL p = .0494, LH p = .0001, CH p = 0.0463).

Infarct size

Motion ability is closely related to the degree of brain injury. The infarct size was evaluated by TTC staining. Compared with the control group, the low-dose supplementation reduced the cerebral infarct volume; however, the high-dose supplementation increased the cerebral infarct volume (Figure 4(a) and (b)).

Figure 4.

Low-dose testosterone supplementation decreased infarct volume after stroke, and high-dose testosterone supplementation increased infarct size. (a) Representative photos of brain stained by TTC. (b) Infarct size (Control 58.42 ± 5.416 vs low 51.05 ± 5.918 vs high 64.25 ± 4.023, seconds, n = 6–8; CL p = .039, LH p = .0003, CH p = .0327).

Inflammation

We tested the inflammatory response of the tissues in the injured site 72 h after stroke. Compared with the control group, low-dose supplementation significantly decreased the mRNA expression of the pro-inflammatory cytokines Tnf-α, Il-6, and Mcp-1, while the high-dose supplementation increased the inflammatory responses (Figure 5(a)–(c)).

Figure 5.

Low-dose testosterone supplementation decreased inflammatory factors after stroke, and high-dose testosterone supplementation increased inflammatory factors. (a) The relative mRNA expression of Tnf-α (n = 6). (b) The relative mRNA expression of Il-6 (n = 6). (c)The relative mRNA expression of Mcp-1 (n = 6).

Oxidative stress

Tissue injury-induced macrophages will increase the production of peroxide products, thereby exacerbating brain injury. We measured oxidative stress in the injury site 72 h after stroke. Compared with the control group, low-dose supplementation decreased MDA and increased T-AOC. On other hand, high-dose supplementation increased MDA and decreased T-AOC (Figure 6(a) and (b)). This indicates that normal supplementation of testosterone can reduce stroke-induced oxidative stress in elderly mice.

Figure 6.

Low-dose testosterone supplementation decreased oxidative stress after stroke, and high-dose testosterone supplementation increased oxidative stress. (a) MDA (control 2.287 ± 0.3108 vs low 1.182 ± 0.1365 vs high 2.696 ± 0.1153, seconds, n = 4; CL p = .0311, LH p = .0001, CH p = .0487). (b) T-AOC (control 0.1546 ± 0.01,186 vs low 0.2288 ± 0.01,371 vs high 0.134 ± 0.01,037, seconds, n = 4; CL p = .0002, LH p = .0001, CH p = .0396).

Discussion

Testosterone has been used as a safe drug for sex hormone supplementation in middle-aged, elderly men, and transgender people.^24,25 Recently, this view is changing, and excessive uptake may increase the incidence of cardiovascular diseases.^25,28 Our study showed that normal supplementation reduced brain injury after stroke by reducing brain inflammation and oxidative stress. Surprisingly, excessive supplementation produced opposite result, aggravating the brain injury after stroke. This study illustrates the value and risk of testosterone from a dose perspective. Therefore, this study confirmed that appropriate supplementation of testosterone is beneficial to reduce the risk of stroke injury in people with below-normal physiological levels of testosterone.

Inflammation and oxidative stress are important causes of tissue injury.^37–41 Stroke increased inflammatory cell aggregation and oxidative stress production in the injury site, which is usually leading to secondary brain injury.^42–44 The secondary injury could be ameliorated by reducing inflammation and oxidative stress. Previous studies have shown that the decreased testosterone levels in the elderly are related to the increased incidence of cardiovascular diseases and are inextricably linked to inflammation and oxidative stress. ²⁹ Testosterone supplementation decreased these symptoms. ⁴⁵ For example, in the new coronavirus (COVID-19) infection, testosterone significantly reduced the patient’s excessive inflammatory response and improves the prognosis. ⁴⁶ In multiple sclerosis, testosterone can protect cultured neurons from glutamate-induced toxicity and oxidative stress. ⁴⁷ In Alzheimer’s, testosterone can protect synapses by reducing the oxidative stress response. ⁴⁸ In rats with severely injured testicular, increasing the serum testosterone level can significantly reduce oxidative injury and promote the expression of inflammatory cytokines. ⁴⁹ These studies show that increasing testosterone reduces oxidative stress and inflammation by stabilizing the body’s antioxidant system and immune system.

However, some studies have pointed out that excessive testosterone supplementation may increase the risk of cardiovascular events.^50,51 These results seem to be contradictory, but they are intrinsically linked. One explanation is that regular testosterone level increases oxidative stress adaptability of cells. This pretreatment method protects cells from injury caused by subsequent exposure to oxidative stress, thereby producing a protective effect. In this theory, pretreatment is a protective process, in which exposure to a slight injury can make cells better able to withstand greater injury that follows. This phenomenon has been observed in a variety of cells. ⁵² It is speculated that excessive testosterone supplementation beyond normal physiological levels may increase inflammation and oxidative stress, exceed cell tolerance, and ultimately lead to injury. Consistent with these results, we also confirmed the importance of testosterone supplementation in preventing the exacerbation of brain injury after stroke in elderly mice, which showed that excessive testosterone supplementation will increase the risk of brain injury after stroke.

This study has some limitations. First, this experiment is to mimic the effects of testosterone therapy in elderly males; however, it is not clarifying the effects of testosterone therapy on young mice. Second, in this study we only used male mice for our experiment. Third, it is not clarifying the safe dose of testosterone therapy. Therefore, in the future, we are planning to use a series of testosterone concentrations to investigate the effects of testosterone on brain injury in elderly mice after stroke. Fourth, the pattern of testosterone use was also a limitation of this study. In terms of dosage, we refer to previous studies; however, other people’s testosterone is usually handled by gavage or daily injections. In the study, it was found that gavage of oil may increase the mortality of mice due to improper operation. Daily injections can cause tissue calcification, which in turn increases systemic inflammatory responses. Therefore, we did a twice-weekly but higher dose injection. It has the potential to affect results through unstable testosterone levels. A mini pump might be a better way. In the future, we will continue to deepen our research in this field. Nevertheless, suitable mice need to be housed for a long time for a detailed study. Further research will focus on the mechanisms by which low-dose supplementation alleviates stroke injury, and the mechanisms by which supplementation at supraphysiological levels exacerbates stroke injury. Also, castrated mice are also very good animal models to study testosterone replacement therapy to study the role and mechanism of testosterone in the future. Finally, the sample size of this study is small, so we only used T-test and Tukey’s test. Power analysis was not used.

Conclusions

In summary, appropriate testosterone supplementation at appropriate levels in elderly mice can alleviate post-stroke injury by reducing inflammation and oxidative stress, but excessive supplementation will aggravate post-stroke injury. Therefore, testosterone replacement therapy can reduce the risk of stroke in patients with insufficient testosterone. This research has important implications for the application of testosterone replacement therapy.