Introduction: An increasing number of middle-aged men are being screened for low testosterone levels and the number of prescriptions for various forms of testosterone replacement therapy (TRT) has increased dramatically over the last 10 years. However, the safety of TRT has come into question with some studies suggesting increased morbidity and mortality. Objective: Because the benefits of estrogen replacement in postmenopausal women and ovariectomized rodents are lost if there is an extended delay between estrogen loss and replacement, we hypothesized that TRT may also be sensitive to delayed replacement. Methods: We compared the effects of testosterone replacement after short-term (2 weeks) and long-term testosterone deprivation (LTTD; 10 weeks) in middle-aged male rats on cerebral ischemia, oxidative stress, and cognitive function. We hypothesized that LTTD would increase oxidative stress levels and abrogate the beneficial effects of TRT. Results: Hypogonadism itself and TRT after short-term castration did not affect stroke outcome compared to intact rats. However, after long-term hypogonadism in middle-aged male Fischer 344 rats, TRT exacerbated the detrimental behavioral effects of experimental focal cerebral ischemia, whereas this detrimental effect was prevented by administration of the free-radical scavenger tempol, suggesting that TRT exacerbates oxidative stress. In contrast, TRT improved cognitive performance in non-stroked rats regardless of the length of hypogonadism. In the Morris water maze, peripheral oxidative stress was highly associated with decreased cognitive ability. Conclusions: Taken together, these data suggest that TRT after long-term hypogonadism can exacerbate functional recovery after focal cerebral ischemia, but in the absence of injury can enhance cognition. Both of these effects are modulated by oxidative stress levels.

The interest in testosterone replacement therapy (TRT) has grown rapidly in the last decade [1, 2]. In the absence of known organic causes of hypogonadism such as pituitary or testicular disease, safety and prescribing practices for younger men remain in question [3]. Because symptoms of “low T” are indistinct and T levels do not fall precipitously in men as they do in women after menopause, the duration, extent, and causes of “low T” in middle-aged men are rarely clear [4]. Currently, contraindications for TRT prescribing include polycythemia (HCT >54%), breast cancer, and prostate cancer [4], untreated severe sleep apnea, and uncontrolled congestive heart failure [5]. Other than these indications, TRT is considered relatively safe, particularly in older men (>65), although the benefits may be limited to mood, bone health, and sexual function [6].

Although it is clear that low T levels are associated with cardiovascular disease, including stroke [7-9], the health effects of TRT are less clear. Most recent longitudinal studies support the benefits of TRT for cardiovascular and cerebrovascular disease risk [10-12]. However, the duration of subclinical hypogonadism for most men is not known, and despite relatively constant levels of low T in laboratory testing, sales for TRT in the US quadrupled between 2000 and 2011 [13]. Moreover, in the US >90% of prescriptions for TRT are for men under 65 years of age [13], and the number of prescriptions for TRT far exceeds the prevalence of strictly diagnosed hypogonadism (2 low fasted morning T levels, at least one additional symptom) [1].

In women, hormone therapy (HT) replacement with estrogens or estrogens + progestins after menopause can have both beneficial and detrimental effects. In addition to predicted adverse effects such as breast cancer, the detrimental effects of HT observed in the Women’s Health Initiative trials included an increased risk of cerebrovascular events, including stroke, thromboembolism, and cognitive impairment [14, 15]. One of the primary hypotheses for the cerebrovascular effects is that the average age of women entering the study (>65 years) meant that treatment was initiated after a significant period of hypogonadism after menopause and, thus, women missed a “critical window” for benefit [16, 17]. This critical window is recapitulated in several laboratory reports regarding the beneficial effects of estrogen in experimental stroke in mice [18] and rats [19] wherein a delay of as little as 10 weeks in estrogen replacement abolishes neuroprotection. Similarly, estrogen is no longer protective in global cerebral ischemia after long-term estrogen deprivation (LTED) [20]. LTED also impairs estrogen-dependent support of immune function [18] and antioxidant activity [20]. Furthermore, although immediate treatment with estrogen after ovariectomy protects the brain from ischemia, LTED increases amyloidogenesis in the hippocampus, and this effect cannot be inhibited by estrogen [21].

We hypothesized that there may be a similar “critical window” for the beneficial effects of TRT. Low T is associated with several comorbid states that impact the response to cerebral ischemia including obesity, hypertension, dyslipidemia, and type 2 diabetes mellitus [22, 23]. One common feature of these low-T-associated states is elevated oxidative stress levels, and the underlying increase in oxidative stress may increase susceptibility to the detrimental effects of TRT. Although T can have antioxidant actions in experimental stroke [24-26], T can also induce oxidative stress in the brain [27]. In addition, preexisting oxidative stress can result in toxic effects of T treatment in dopaminergic neurons [28] and male rat brains [29].

The effects of T on stroke outcome in males appear to be age dependent with studies of short-term T deprivation and replacement revealing beneficial effects in middle age, but detrimental effects in young animals [30-34]. Supraphysiological T or DHT leads to worse ischemic injury [31-33, 35], but low doses of either T or DHT have also been shown to improve stroke outcome in young animals [33, 35].

As with experimental stroke, several studies support a correlation between T levels and cognition. Longitudinal studies show that men with the greatest decline in T levels with aging have more cognitive decline and greater Alz-heimer’s disease risk [36, 37]. Furthermore, reductions in T to treat prostate cancer increases stroke risk in humans [38, 39]. Supraphysiological TRT can enhance visuospatial cognition in older men in some studies [40-42]. However, the data in human studies are inconsistent, with several studies showing no benefits [43-46], although TRT effects may be dose dependent [40, 47].

In this study, we used physiological TRT in middle-aged rats to determine whether a critical window exists for the effects of testosterone on stroke and cognition and whether oxidative stress plays a role in the beneficial and/or detrimental effects of TRT.

Animals

All protocols were approved by the Institutional Animal Care and Use Committee at the University of North Texas Health Science Center and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Middle-aged (12 months) male Fischer 344 rats were obtained from the National Institutes on Aging colony. Rats were maintained on a 12:12 light:dark cycle (lights on 7:00 a.m.) with ad libitum rat chow and water in an AAALAC-approved centralized animal facility. Animals were weighed weekly.

Gonadectomy and Hormone Treatment

Rats were gonadectomized or sham gonadectomized under isoflurane anesthesia (2–2.5%). Sham surgery consisted of an incision in the scrotum and visualization of the testes. Testosterone or cholesterol treatments were performed with subcutaneous silastic capsules. Crystalized testosterone and cholesterol were obtained from Steraloids (Newport, RI) and packed into silastic tubing (1.47 mm i.d. × 1.96 mm o.d. × 10 mm length, Dow Corning, Midland, MI) sealed on the ends with silastic adhesive. Capsules were placed in 10× PBS overnight to ensure patency (floating capsules were used). Two capsules were placed in a subcutaneous pocket in the chest under brief isoflurane anesthesia.

Focal Cerebral Ischemia

Rats were randomly assigned to 5 experimental groups (Fig. 1). Intact rats were sham gonadectomized and otherwise untreated throughout the protocol. Long-term testosterone-deprived (LTTD) rats were castrated 10 weeks prior implantation of cholesterol (LTTD) or testosterone containing implants (LTTD + T). A third group of LTTD rats was treated with testosterone and the antioxidant Tempol (1 mg/mL) in their drinking water starting 8 weeks after castration and continuing until the conclusion of the study. Short-term testosterone deprived with testosterone (STTD + T) were castrated and treated 2 weeks later with T capsules. Three weeks after treatments began, rats underwent middle cerebral artery occlusion (MCAO) as described below. All treatments were continued throughout behavioral studies and until rats were humanely euthanized.

Fig. 1.

Experimental design for stroke studies. Five groups were used in the stroke study, gonad-intact rats (Intact) with cholesterol implants (C), LTTD castrated for 10 weeks prior to cholesterol implants (LTTD), LTTD + T rats castrated (X) for 10 weeks prior to testosterone (T) implants, and STTD rats castrated for 2 weeks prior to testosterone (STTD + T). A fifth group that underwent LTTD + T but also began treatment with TEMPOL 2 weeks prior to T treatment. MCAO surgery is indicated with arrows. Behavior tests were performed for the following 2 weeks. MCAO, middle cerebral artery occlusion; LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Fig. 1.

Experimental design for stroke studies. Five groups were used in the stroke study, gonad-intact rats (Intact) with cholesterol implants (C), LTTD castrated for 10 weeks prior to cholesterol implants (LTTD), LTTD + T rats castrated (X) for 10 weeks prior to testosterone (T) implants, and STTD rats castrated for 2 weeks prior to testosterone (STTD + T). A fifth group that underwent LTTD + T but also began treatment with TEMPOL 2 weeks prior to T treatment. MCAO surgery is indicated with arrows. Behavior tests were performed for the following 2 weeks. MCAO, middle cerebral artery occlusion; LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

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Middle Cerebral Artery Occlusion

Rats underwent MCAO via intraparenchymal injection of endothelin 1 (1.5 µg in 3 µL PBS; Sigma) adjacent to the left middle cerebral artery, as previously described [48]. Briefly, rats were anesthetized with isoflurane and placed in a stereotax with Bregma and Lambda level. Body temperature was maintained with a servo-controlled heating pad connected to a rectal thermometer. After opening the scalp through a midline incision, a laser Doppler flow (LDF) probe (Perimed) was attached to the left side of the skull 3 mm caudal and 5 mm lateral to Bregma to assess parietal cortex blood perfusion. A burr hole was drilled in the skull 0.9 mm rostral and 3.4 mm lateral to Bregma on the left side. A 26-gauge Hamilton syringe attached to a stereotaxically mounted Micro4 syringe pump (World Precision Instruments) was lowered 8.5 mm from Bregma to inject endothelin 1 (ET-1) over a period of 6 min. The syringe was left in place 3 min before removal. Bone wax was used to close the skull. The scalp was infiltrated with 0.25% bupivacaine (Hospira, Lake Forest, IL, USA), and the scalp was closed with surgical staples and treated with triple antibiotic ointment. A group of intact sham stroke rats was injected with PBS vehicle instead of ET-1. The surgeon was blinded to treatment.

Seven rats were euthanized after tumors were detected. Four rats died at 4, 5, 6, and 7 weeks following gonadectomy, and 3 rats died within 3 days of stroke surgery.

Behavioral Assessment of Rats Following Ischemia

After 3, 7, and 14 days, MCAO rats underwent neurological testing including an overall neurological score, the cylinder test, rotarod, and ladder walking. All tests were carried out in a room lit with red lights between 2:00 and 5:00 p.m. The neurological score used an 11-point scale similar to a 14-point scale used previously [49]. The composite score is derived from the following: spontaneous circling (0 = no circling to 3 = continuous ipsilateral circling), contralateral hindlimb retraction (0 = immediate replacement to 3 = no replacement of >2 min), bilateral forelimb grasp (0 = grabs with all digits on both forelimbs to 3 = cannot grasp with either forelimb), and contralateral forelimb flexion (0 = both limbs extend when lifted to 2 = shoulder adduction with forelimb flexion).

The cylinder test for forelimb placement was performed as previously described [49] and scored by video analysis by a blinded observer. Assessments were performed on the day prior to MCAO (day –1) and post-stroke days 3, 7, and 14. Rats were placed in a clear plexiglass cylinder for 10 min and forelimb touches on the cylinder side when rearing was recorded. Data are expressed as a ratio of contralateral to ipsilateral touches.

An accelerating Rotarod (Omnitech Electronics, Columbus, OH, USA) was used to assess balance and motor learning. Rats were trained in 4 trials/day for 3 days prior to MCAO as the Rotarod accelerated from 0 to 75 rpm in 150 s. The time to fall on the final day of training, 1 day before MCAO, was recorded as the baseline ability. Trials were repeated on days 3, 7, and 14 following MCAO. Data are reported as the average of 4 daily trials.

Three days prior to stroke, rats were acclimated to a ladder walking test on a 4-foot long automated horizontal plexiglass ladder with rungs spaced 0.5 inches apart (San Diego Instruments, CA, USA). The ladder was suspended 2 feet above a table. Rats were placed on one end of the ladder and encouraged to traverse the ladder to a dark box by air puffs and a bright spotlight. An infrared beam-activated timer automatically started when rats moved to the first rung and turned off when rats reached the box. A separate infrared beam detected foot faults that were verified by video recording. To account for differences in motivation and speed, foot faults are reported as a function of total time to cross the ladder.

Cognitive Experimental Protocol

A separate cohort of animals was randomly assigned to 4 groups. Intact rats were untreated throughout the protocol. LTTD rats were castrated 10 weeks prior implantation of cholesterol (LTTD) or testosterone containing implants (LTTD + T). STTD + T were castrated and treated immediately with T capsules. All treatments were continued throughout behavioral studies and until rats were humanely euthanized.

Cognitive Assessment

Rats were assessed for cognitive function using a Y-maze, an object location memory test (OLMT), and Morris water maze (MWM) starting 12 weeks after randomization. The Y-maze was used to assess hippocampal-dependent memory [50] in a black plexiglass maze with 3 identical arms (50 cm L × 13 cm W × 35 cm H). Rats were placed in one arm of the Y-maze facing the closed end. A poster on the wall adjacent to the arms of the maze was used as an external spatial cue. Rats were allowed to explore the maze stem and a single open arm for 15 min during an informational trial. Twenty-four hours later both arms were open, and rats were allowed to explore the maze for 5 min. The number of entries and amount of time spent in each arm were determined using video recording with AnyMaze with a camera positioned above the maze. Total exploration time and percent time in the familiar and novel arms were calculated.

The OLMT was performed as described by McConnell et al. [51]. Rats underwent 4 days of habituation to an 80 × 80 × 30 cm open field with a black floor and white plexiglass walls. Days 1 and 2 consisted of 20 min habituation in pairs. On day 1, the apparatus contained bedding material that was removed on day 2. On days 3 and 4, habituation occurred individually, again with bedding present on day 3, but not on day 4. The following day rats underwent an exposure trial with 2 identical objects (plastic juice containers) placed 20 cm from the walls. A poster was placed on the wall as an external maze cue. Rats were allowed to explore for 5 min and were recorded with AnyMaze software (Stoelting). The field and objects were washed with 70% ethanol. One object was then moved to one of 2 new locations (counterbalanced), and the test was repeated after a 30-min delay. The same procedure was repeated 4 days later, and data from the 2 days were averaged. Exploration was defined as the rat nose falling within 2 cm of the object.

The MWM was performed as previously described [52]. Briefly, after training in a straight maze with visible platform (3 trials × 2 sessions), rats underwent 7 MWM sessions beginning the following day. Four days of acquisition trials (3 trials, 10 min intertrial interval) were followed by a 2-day rest. A retention session was performed on the day following the rest period (3 trials, 10 min intertrial interval). On the following 2 days, rats underwent reversal trials in which the hidden platform was moved to a new location. Probe trials without the platform were run prior to acquisition session on day 4 and 10 min after the final reversal trial on day 7. Data were collected by a digital camera and AnyMaze software. Path length (PL), latency to find the platform, and swim speed were automatically calculated. A group of young (3 months) intact rats was added to the MWM protocol for comparison.

Tissue Collection

Within 5 days of the conclusion of behavioral testing, rats were deeply anesthetized with isoflurane and rapidly decapitated with a guillotine. Rats were euthanized between 10:00 a.m. and 12:00 p.m. Trunk blood was collected into EDTA-coated tubes, inverted multiple times, and placed on ice for <60 min. Blood was centrifuged for 15 min at 2,000 g, and plasma was collected and frozen at –80°C until assay. Brains were removed, cooled in ice-cold saline for 2 min, and placed in a brain matrix. A 2 mm coronal section was made between –0.5 and –2.5 mm relative to Bregma. The remaining forebrain and hindbrain were fixed for 48 h in 4% formaldehyde at 4°C before being moved to PBS for storage before cutting.

Plasma Measurements

Total testosterone concentrations were measured in duplicate from plasma following euthanasia using a commercial ELISA from BioVendor (RTC001R, Ashville, NC, USA). Peripheral oxidative stress was measured in plasma with a commercial kit for advanced oxidation protein products (AOPP; OxiSelect, Cell Biolabs, Inc., San Diego, CA, USA) using manufacturer’s instructions. Samples were diluted 1:6 and assayed in duplicate. AOPP results from the interaction of reactive oxygen species with proteins to yield modifications including dityrosine, pentosidine, and carbonyls [53]. Increased AOPP levels are associated with many pro-inflammatory diseases and are used as a biomarker for oxidative stress [54].

Statistical Analysis

All data were analyzed with GraphPad Prism version 8. For biochemical data (T, AOPP, western blotting), results were analyzed by one-way analysis of variance (ANOVA) with treatment group as the independent variable and p set as <0.05. Pairwise comparisons were made with Tukey-Kramer tests. For cognitive testing, 2-way ANOVAs were used with treatment group and time as independent variables. In some cases, repeated-measures ANOVAs used a mixed-effect model due to missing values. Pairwise comparisons were made with Tukey-Kramer tests. In some cases with pretreatment measures, a Dunnett’s test was also used to compare baseline to post stroke results. Covariance was determined with Pearson’s correlation coefficient. For post MCAO behavioral assessments, data were analyzed by 2-way ANOVA with repeated measures with treatment group and time as the independent variables and p set at 0.05. Pairwise comparisons were made with Tukey-Kramer tests, and comparisons to baseline measures were made with Dunnett’s tests. All data are presented as mean ± SD, and animal numbers are stated in the figure legends. Rats that did not complete all days of testing for a particular behavioral assessment due to incapacitation, death, failure to meet pretest criteria, or failure to perform test were excluded from repeated-measures analysis resulting in different n’s across experiments.

Intact and sham stroke rats gained significant weight over the initial 12 weeks prior to stroke (11.3 and 40.1 g, respectively, p < 0.05). Although other treatment groups gained weight, the changes were not significant (data not shown). In non-stroked rats from the cognition study cohort, LTTD significantly (F3,35 = 7.65; p < 0.001) reduced circulating total testosterone to near undetectable, and silastic implants of crystalline testosterone restored levels to those observed in age-matched testes-intact rats (Fig. 2a). Also, in non-stroke rats, peripheral oxidative stress, as measured with AOPP, did not differ in intact and LTTD rats (Fig. 2b). Testosterone treatment after LTTD did not significantly alter AOPP, but T significantly reduced AOPP levels in STTD + T rats (ANOVA F3,34 = 5.08, p < 0.006, Tukey, p > 0.05; Fig. 2b).

Fig. 2.

Testosterone and AOPP levels in plasma. a LTTD (dark shaded bars) rats had significantly (* p < 0.05) lower plasma total testosterone levels than all other groups (p > 0.01). T implants (LTTD + T, light shaded bars, and STTD + T, hatched bars) restored T to Intact levels (n = 9–11 per group). b STTD + T rats had significantly (p < 0.05) lower plasma levels of AOPP than both Intact and LTTD rats, but LTTD + T were not different from any other groups (n = 7–11 per group). AOPP, advanced oxidation protein products; LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Fig. 2.

Testosterone and AOPP levels in plasma. a LTTD (dark shaded bars) rats had significantly (* p < 0.05) lower plasma total testosterone levels than all other groups (p > 0.01). T implants (LTTD + T, light shaded bars, and STTD + T, hatched bars) restored T to Intact levels (n = 9–11 per group). b STTD + T rats had significantly (p < 0.05) lower plasma levels of AOPP than both Intact and LTTD rats, but LTTD + T were not different from any other groups (n = 7–11 per group). AOPP, advanced oxidation protein products; LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Close modal

Injection of ET-1 significantly reduced parietal LDF, indicating successful constriction of the middle cerebral artery (Fig. 3). In response to vehicle injection, LDF in the parietal cortex increased by 19% (Fig. 3). In contrast, ET-1 injection (1.5 µg) resulted in significantly decreased LDF (F5,83 = 74.3; p < 0.001). In Intact rats, the drop averaged 42%, but LDF fell significantly (Tukey df 83, p < 0.05) further in all castrate experimental groups (Fig. 3). However, the drop was not different among the treatment groups, indicating that differences among these groups are not a result of differential ischemia.

Fig. 3.

LDF following endothelin 1 injection. Change in parietal LDF following injection of vehicle (Sham) or ET1 adjacent to the middle cerebral artery. ET1 significantly reduced LDF in all groups compared to sham (* p < 0.05), and all castrate groups (LTTD, LTTD + T, TEMPOL, STTD + T) were significantly different (# p < 0.05) than the Intact group. n = 13–20 per group. LDF, laser Doppler flow; LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Fig. 3.

LDF following endothelin 1 injection. Change in parietal LDF following injection of vehicle (Sham) or ET1 adjacent to the middle cerebral artery. ET1 significantly reduced LDF in all groups compared to sham (* p < 0.05), and all castrate groups (LTTD, LTTD + T, TEMPOL, STTD + T) were significantly different (# p < 0.05) than the Intact group. n = 13–20 per group. LDF, laser Doppler flow; LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Close modal

Neurological deficits were present in all stroke groups 3, 7, and 14 days after stroke (Fig. 4). Two-way repeated-measures ANOVA revealed main effects of time (F1.859, 158 = 18.63; p < 0.001), group (F15, 85 = 18.28; p < 0.001), and a time × group interaction (F10, 170 = 3.91; p < 0.001). LTTD rats treated with testosterone showed no improvement over time and exhibited a significantly worse neuroscore than all the other treatment groups (Tukey, p < 0.01; Fig. 4). Multiple comparisons revealed that testosterone treatment was detrimental when treatment was delayed 10 weeks (LTTD + T), but not 2 weeks (STTD + T; Fig. 4). The detrimental effect in the LTTD + T rats was abrogated by treatment with the antioxidant Tempol (Fig. 4).

Fig. 4.

Neurological deficit scores after MCAO. Neurological deficit scores (Neuroscore) on days 3, 7, and 14 after MCAO (mean ± SE, n = 12–20 per group). All stroke groups showed significant deficits compared to sham (p < 0.001), and LTTD + T was significantly different from all other groups (* p < 0.001). On days 3, 7, and 14, Tempol-treated LTTD + T rats performed significantly better than LTTD + T rats (p < 0.01). STTD + T rats were signif-icantly improved compared to LTTD + T rats on days 7 and 14 (p < 0.01), and LTTD rats were significantly improved compared to LTTD + T rats on day 14 (p < 0.01). LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Fig. 4.

Neurological deficit scores after MCAO. Neurological deficit scores (Neuroscore) on days 3, 7, and 14 after MCAO (mean ± SE, n = 12–20 per group). All stroke groups showed significant deficits compared to sham (p < 0.001), and LTTD + T was significantly different from all other groups (* p < 0.001). On days 3, 7, and 14, Tempol-treated LTTD + T rats performed significantly better than LTTD + T rats (p < 0.01). STTD + T rats were signif-icantly improved compared to LTTD + T rats on days 7 and 14 (p < 0.01), and LTTD rats were significantly improved compared to LTTD + T rats on day 14 (p < 0.01). LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Close modal

Analysis of the cylinder test was complicated by the number of animals that failed to rear at all following stroke (Fig. 5a) with fewer animals rearing in all but the Sham group by day 14. Because not all rats reared on each day, a mixed-effect 2-way ANOVA was used for analysis. A significant effect of time (F2.698, 158.3 = 5.92; p = 0.002) and time × treatment interaction (F15, 176 = 2.26; p < 0.007) was observed, but no significant treatment effect was observed. Dunnett’s multiple comparisons showed that, compared to pre-stroke (day –1) results, STTD + T was significantly biased toward the ipsilateral forelimb on day 3 (Dunnett, p = 0.002) and LTTD + T was significantly biased on day 14 (Dunnett, p = 0.001) after MCAO (Fig. 5b).

Fig. 5.

Forelimb bias in the cylinder test after MCAO. a The number of rats per group that displayed rearing decreased over time in all stroke groups. b Forelimb bias in the cylinder test showed a significant effect of time (F2.698, 158.3 = 5.92; p < 0.002) and time × treatment interaction (F15,176 = 2.26; p < 0.007) was observed, and Dunnett’s multiple comparisons showed that, compared to pre-stroke results, STTD + T was significantly biased toward the ipsilateral forelimb on day 3 (* Dunnett, p < 0.002) and LTTD + T was significantly biased on day 14 (* Dunnett, p < 0.001). LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Fig. 5.

Forelimb bias in the cylinder test after MCAO. a The number of rats per group that displayed rearing decreased over time in all stroke groups. b Forelimb bias in the cylinder test showed a significant effect of time (F2.698, 158.3 = 5.92; p < 0.002) and time × treatment interaction (F15,176 = 2.26; p < 0.007) was observed, and Dunnett’s multiple comparisons showed that, compared to pre-stroke results, STTD + T was significantly biased toward the ipsilateral forelimb on day 3 (* Dunnett, p < 0.002) and LTTD + T was significantly biased on day 14 (* Dunnett, p < 0.001). LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Close modal

The aged Fisher 344 rats performed poorly on the rotarod, falling within seconds of the start of rotation. However, all groups reached the same competency by the day prior to stroke (Fig. 6). Similar to neurological deficit scores, ANOVA revealed significant main effects of time (F2.248, 69.67 = 224.7; p < 0.001), treatment (F4, 31 = 217; p < 0.001), and time × treatment interaction (F12, 93 = 2.26; p < 0.001). The LTTD + T group performed significantly worse than all other groups (Tukey p < 0.05). On day 3, STTD + T performed significantly better than other stroked groups (Tukey p < 0.001; Fig. 6). By day 7, all groups began to improve except the LTTD + T group, whose performance continued to decline through day 14 (Fig. 6). One squad of Tempol-treated animals (n = 4) was later added to assess the effect of reducing oxidative stress. Tempol significantly reversed the detrimental effects of T in the LTTD + T group on days 7 and 14 (Tukey p < 0.01; Fig. 6).

Fig. 6.

Latency to fall from accelerating rotarod after stroke. The latency to fall (s) was similar in all groups prior to stroke (day –1) and decreased by day 3 after stroke. A significant effect of time, treatment, and interaction was observed (p < 0.001, n = 5–14 per group). All but the LTTD + T group showed some improvement over time such that the LTTD + T group was significantly different from all other treatments (* p < 0.001). Comparisons for each day showed that by day 3, the STTD + T group improved relative to the other stroke groups (†), and LTTD also improved by day 7. However, on days 7 and 14, the LTTD + T group performed significantly worse than all other groups. LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Fig. 6.

Latency to fall from accelerating rotarod after stroke. The latency to fall (s) was similar in all groups prior to stroke (day –1) and decreased by day 3 after stroke. A significant effect of time, treatment, and interaction was observed (p < 0.001, n = 5–14 per group). All but the LTTD + T group showed some improvement over time such that the LTTD + T group was significantly different from all other treatments (* p < 0.001). Comparisons for each day showed that by day 3, the STTD + T group improved relative to the other stroke groups (†), and LTTD also improved by day 7. However, on days 7 and 14, the LTTD + T group performed significantly worse than all other groups. LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Close modal

In the ladder walking test, there was a significant effect of treatment (F6, 60 = 8.37, p < 0.001) and a treatment by time interaction (F10, 120 = 2.81, p < 0.004), but no significant effect of time alone. All stroke groups performed significantly worse than sham stroke rats (Tukey, p < 0.05; Fig. 7). There was also a significant difference between the LTTD and the LTTD + T group (Tukey, p = 0.016), with testosterone treatment leading to progressively worse performance (Fig. 7). This effect was significant on day 14, when the LTTD + T group had significantly more foot faults than the LTTD group (Tukey, p < 0.003). As with other behavioral assessments, Tempol reversed the detrimental effect of testosterone in the LTTD + T group (Tukey, p < 0.04).

Fig. 7.

Performance on horizontal challenge ladder after stroke. All stroke groups had more foot faults on the challenge ladder than Sham rats (* p < 0.001, n = 7–13 per group), and there was a significant overall difference between the LTTD and LTTD + T groups (# p < 0.05). LTTD, LTTD + T, and Tempol groups were significantly impaired on day 7 ( p < 0.05). On day 14, all groups were impaired compared to the Sham group, but the LTTD and LTTD + T groups also differed from one another (# p < 0.05). LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Fig. 7.

Performance on horizontal challenge ladder after stroke. All stroke groups had more foot faults on the challenge ladder than Sham rats (* p < 0.001, n = 7–13 per group), and there was a significant overall difference between the LTTD and LTTD + T groups (# p < 0.05). LTTD, LTTD + T, and Tempol groups were significantly impaired on day 7 ( p < 0.05). On day 14, all groups were impaired compared to the Sham group, but the LTTD and LTTD + T groups also differed from one another (# p < 0.05). LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Close modal

Three different tasks were used to assess the effects of hormone deprivation and T replacement on cognitive function. In the Y-maze, there were no significant dif-ferences among the groups in novel arm exploration (Fig. 8a). In the OLMT, there was a significant overall effect of object location, with all groups showing increased exploration of the moved object (F1, 39 = 15.94, p = 0.003), but no treatment effects were observed (Fig. 8b). To ensure that rats were cognitively aged, a group of four 3-month-old males were added for the MWM. The MWM revealed that exogenous testosterone improved performance compared to intact rats regardless of the timing of T (Fig. 9). Data in Figure 9a, c, and e are expressed as a mean of acquisition sessions 2–4. One-way ANOVA for PL showed a significant effect of treatment (F4,39 = 8.15, p < 0.001). Young rats had a shorter PL than both Intact and LTTD rats (Tukey p < 0.05), and treatment of castrated old rats with exogenous T improved performance compared to intact rats regardless of treatment timing (Tukey p < 0.01; Fig. 9a, b). A similar main effect of treatment was observed for latency to find the platform (F4,39 = 4.49, p < 0.005; Fig. 9c, d), and both T treated groups showed significant improvement from intact rats (Tukey p < 0.05). Interestingly, LTTD was not different from intact or T-treated groups despite the observation that T levels were equivalent in the Intact and T-treated groups (Fig. 2). Young rats swam significantly faster than aged rats (F4,39 = 11.67, p < 0.001; Fig. 9e, f), but no differences were observed in the treatment groups. Pearson correlations demonstrated a strong relationship between AOPP levels and increased MWM PL (R2 = 0.337, p < 0.002), and a small, but significant relationship between T and AOPP, with higher AOPP associated with low T levels (Fig. 10). No similar relationship between T and PL was observed.

Fig. 8.

Cognitive performance on Y-maze and OLMT. a No significant differences among treatment groups were observed in the Y-maze. b There was a significant main effect (p < 0.001) of object location in the OLMT in which rats in all groups explored the moved object more than the unmoved object. No treatment group differences were observed (n = 9–12 per group). LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Fig. 8.

Cognitive performance on Y-maze and OLMT. a No significant differences among treatment groups were observed in the Y-maze. b There was a significant main effect (p < 0.001) of object location in the OLMT in which rats in all groups explored the moved object more than the unmoved object. No treatment group differences were observed (n = 9–12 per group). LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Close modal
Fig. 9.

Exogenous testosterone improves MWM performance. a Mean ± SD overall path length (PL) for the Acquisition phase indicates a significant improvement from intact rats in the LTTD + T and STTD + T groups (* p < 0.01 vs. Intact), but all aged animals performed worse than young intact rats (# p < 0.05 vs. Young). b Mean ± SD PL (cm) to find the hidden platform in the Acquisition, Retention, and Reversal phases of the MWM (n = 10 per group). c Learning Index of Latency for the Acquisition phase indicates a significant improvement from Intact rats in the LTTD + T and STTD + T groups (* p < 0.01 vs. Intact). The LTTD group did not differ from any other group. d Mean ± SD of Latency (s) to find the hidden platform in the Acquisition, Retention, and -Reversal phases of the MWM (n = 10 per group). e Mean ± SD Swim Speed during the MWM showed a significant main effect of age with all older groups swimming faster than young intact rats (#p < 0.05), but there were no significant differences among the groups (Tukey-Kramer p > 0.05). LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone. f Mean ± SD of Swim Speed (cm/s) to find the hidden platform in the Acquisition, Retention, and Reversal phases of the MWM (n = 10 per group). LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Fig. 9.

Exogenous testosterone improves MWM performance. a Mean ± SD overall path length (PL) for the Acquisition phase indicates a significant improvement from intact rats in the LTTD + T and STTD + T groups (* p < 0.01 vs. Intact), but all aged animals performed worse than young intact rats (# p < 0.05 vs. Young). b Mean ± SD PL (cm) to find the hidden platform in the Acquisition, Retention, and Reversal phases of the MWM (n = 10 per group). c Learning Index of Latency for the Acquisition phase indicates a significant improvement from Intact rats in the LTTD + T and STTD + T groups (* p < 0.01 vs. Intact). The LTTD group did not differ from any other group. d Mean ± SD of Latency (s) to find the hidden platform in the Acquisition, Retention, and -Reversal phases of the MWM (n = 10 per group). e Mean ± SD Swim Speed during the MWM showed a significant main effect of age with all older groups swimming faster than young intact rats (#p < 0.05), but there were no significant differences among the groups (Tukey-Kramer p > 0.05). LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone. f Mean ± SD of Swim Speed (cm/s) to find the hidden platform in the Acquisition, Retention, and Reversal phases of the MWM (n = 10 per group). LTTD, long-term testosterone-deprived rats; STTD + T, short-term testosterone deprived with testosterone.

Close modal
Fig. 10.

Relationships between T, oxidative stress, and cognitive performance. Pearson correlations revealed a significant, but small positive correlation between plasma T and AOPP levels. There was no significant correlation between MWM PL and T, but a significant correlation (R2 = 0.337, p = 0.002) was found between AOPP and PL such that higher oxidative stress correlated with longer paths to the hidden platform in the MWM. MWM, Morris water maze; AOPP, advanced oxidation protein products.

Fig. 10.

Relationships between T, oxidative stress, and cognitive performance. Pearson correlations revealed a significant, but small positive correlation between plasma T and AOPP levels. There was no significant correlation between MWM PL and T, but a significant correlation (R2 = 0.337, p = 0.002) was found between AOPP and PL such that higher oxidative stress correlated with longer paths to the hidden platform in the MWM. MWM, Morris water maze; AOPP, advanced oxidation protein products.

Close modal

Both hypogonadism and testosterone treatment in men are associated with cardiovascular, cerebrovascular, and cognitive dysfunction [55]. Because the beneficial neuroprotective effects of estrogen therapy are sensitive to the length of pretreatment hypogonadism in women and animal models [56, 57], we hypothesized that the -effects of testosterone on the male brain would be lost -following long-term hypogonadism. The present study revealed significantly worse behavioral outcomes after stroke when TRT was delayed 10, but not 2 weeks, after castration in early middle-aged male rats. This detrimental effect occurred at physiological levels of T that were similar to testes-intact rats of the same age. After stroke, long-term hypogonadism itself had little to no effect compared to gonad-intact rats but sensitized the brain to the detrimental effects of TRT. In contrast, TRT after short-term hypogonadism was not different from intact rats with similar circulating T levels. Thus, it appears that the combination of LTTD with T, rather than T itself, resulted in poor outcomes. The detrimental effect of TRT could be completely abrogated with the free radical scavenger tempol, implicating increased oxidative stress as a mechanism for increasing brain sensitivity to TRT. This sensitizing effect was not reflected in AOPP as a measure of peripheral oxidative stress. AOPP was not elevated by LTTD and was only reduced by T after short-term castration, suggesting that the effects of the antioxidant occur in the CNS.

In contrast to stroke, TRT improved performance in the MWM, but not 2 other cognitive tasks, compared to gonadally intact rats regardless of the timing of TRT. Water maze performance was significantly correlated with peripheral oxidative stress, further implicating oxidative stress as a correlate of cognitive decline. Thus, like estrogen replacement in chronically hypogonadal females, chronic hypogonadism in males enhances the detrimental effects of TRT, and this may involve enhanced oxidative stress. In contrast, exogenous T may provide cognitive benefits beyond those observed with endogenous T in middle-aged males. Notably, the effects of exogenous T occurred in the absence of not only testicular androgens, but also testicular peptides such as inhibin and activin. These chronic effects of castration and T on cognition do not appear to be reflected under stroke conditions in the brain itself where oxidative stress levels are expected to be exacerbated.

Hypogonadism in men is associated with cerebrovascular disease, cardiovascular disease, and cognitive decline [58]. Recent studies support benefits of TRT in aged men (>60) including for cardiovascular and cerebrovascular disease [6, 10-12]. Low T, even within the reference range, is associated with an increased risk of ischemic stroke [7, 9, 59-61], and in some studies, TRT reduced risk [11, 12]. Nevertheless, others found that initiation of TRT could increase the risk of major cardiovascular events, including stroke [59, 62]. These studies point to a complex relationship between T levels and cardiovascular disease that may be both time- and dose-dependent [8, 63], resulting in uncertainty in meta-analyses [64] similar to that resulting from reviews of HT in postmenopausal women [56].

The effects of T and hypogonadism on stroke in rodents are inconclusive. In young rats, one week of castration reduced infarct size, and T reversed this effect [30, 65]. Cheng et al. [31] also reported beneficial effects of castration and detrimental effects of supraphysiological doses of T or DHT. Dose effects were confirmed in mice undergoing cardiac arrest/global ischemia [35] or MCAO [33] with lower T doses showing some protective effects and higher doses worsening outcomes. Interestingly, both beneficial and detrimental effects are inhibited by the androgen receptor antagonist flutamide [33]. Only one other study by Cheng et al. [32] confirmed previous studies in young rats showing that castration is neuroprotective, and T reverses this effect. In contrast, T reduced infarct size in 14-month-old rats and 12-month-old mice [32]. However, free T levels were 1.7–40 times the levels in intact animals, suggesting supraphysiological replacement [32]. These results support the notion that high T is detrimental to young animals, but beneficial to older animals. In the present study, total T levels were maintained at the physiological level of the intact middle-aged males, which represents a low physiological dose of T. In addition, the Cheng study used commercial dissolvable pellets (Innovative Research of America), whereas we used silastic capsules. Previous studies demonstrate significant kinetic and supraphysiological dose release from commercial pellets, albeit for estradiol, that could affect outcomes [66-68]. Importantly, in our study, intact, STTD + T, and LTTD + T all achieved similar circulating T, suggesting that the duration of deprivation rather than the dose of T contributed to the detrimental effects in LTTD + T rats.

Contrary to the beneficial effects of T in middle-aged rats and mice [32], we saw neither a detrimental effect of castration nor a beneficial effect of T after short-term castration. Whereas previous studies used 1- to 2-week castration protocols and short-term outcome measures (24–72 h), our study examined functional outcomes for 2 weeks following stroke. Thus, it is possible that the detrimental effects of T are a reflection of the presence of T during the chronic, rather than acute, phase of stroke. However, Fanaei et al. [25] observed beneficial effects of T in young castrate rats when initiated 24 h after focal ischemia, and Pan et al. [69] observed mild behavioral improvements when T treatment began 1 week after stroke. These results would argue for a beneficial effect of T when absent in the acute phase of stroke and present in the chronic phase. Notably, both of these studies used young animals after short-term castration, and we did observe a small benefit in STTD + T rats in rotarod behavior.

The ability of tempol to reverse the detrimental effects of T in long-term castrate rats strongly implicates an interaction between oxidative stress and T in stroke outcomes. Oxidative stress has long been recognized as an important contributor to ischemic injury [70], and antioxidants and free radical scavengers can reduce experimental stroke injury [71]. The ability of tempol to reduce the detrimental effects of T in LTTD rats supports the idea that T exacerbates oxidative stress injury. However, in the absence of ischemic insult, there was no exacerbation of peripheral oxidative stress with LTTD as determined by AOPP and the relationship between T and AOPP, though significance was mild. T moderately reduced AOPP, but only after short-term deprivation, whereas T after long-term deprivation showed an intermediate effect. This difference persisted in spite of similar circulating T levels, suggesting that the level of T alone is not the primary determinant of peripheral oxidative stress. Based on our previous study [72], we hypothesize that this may be the result of a ceiling effect of age on AOPP levels in otherwise healthy animals. In the rodent brain, long-term castration leads to an increase in oxidative stress and antioxidant defenses in several regions including the hippocampus, and TRT can reverse this effect [27, 73, 74]. However, T itself can also act as a mild oxidative stressor [75], and this effect appears to follow an inverted U dose-response, with both low and high doses increasing oxidative stress [27]. Synthetic androgens also increase oxidative stress in the rat brain [76], and in vitro, the effects of T on oxidative stress are also time-dependent [28]. The specific pathways mediating the interaction between T and ROS in the ischemic brain remain to be determined, but our previous studies suggest that NADPH oxidase may play a key role in the detrimental effects of T [29, 77].

The effects of T on water maze performance in non-stroked rats support beneficial actions on hippocampal function that persist even in the context of chronic hypogonadism. Although we observed no detrimental effects of hypogonadism or T in OLMT or the Y maze in the present study, other cognitive tests can reveal steroid-dependent differences. In young rats, 12 weeks of hypogonadism leads to impaired water maze and novel object recognition performance that can be improved with T [78, 79]. Four weeks of hypogonadism can lead to deficits in Barnes maze and Y-maze performance in young male rats that also can be reversed with T [50, 80]. In contrast, Frye et al. [81] showed that short-term gonadectomy impaired water maze performance in 4-month-old Fischer 344 rats but not 13-month-old animals that already had reduced cognitive ability. Moreover, T improved performance in the young, but not middle-aged animals [81]. In our study, we observed a similar increase in PL as rats aged and no significant effect of gonadectomy in middle-aged animals. However, exogenous T improved cognition in both short- and long-term hypogonadal animals. The fact that total T levels in the treated animals were the same as intact rats further suggests that the benefits of T may be due to differences in the metabolism of T in castrate rats or the expression of steroid receptors. Since both short- and long-term castrated rats showed similar benefits after T treatment, the cognitive response, unlike the stroke response, is not refractory to T even after chronic hypogonadism. Another potential reason for this difference is that other testicular factors may inhibit the response of the brain to endogenous T, but this inhibition is removed by castration. For example, castration reduces not only T, but other testicular hormones such as inhibin B and activin that can result in increased follicle-stimulating hormone and luteinizing hormone and may have independent effects on cognition [82]. Although luteinizing hormone and follicle-stimulating hormone were not measured in the current study, castration is expected to increase gonadotropin levels and T treatment is expected to suppress gonadotropins. While there are some data in rodents and humans relating elevated gonadotropins to cognitive decline, it is primarily from studies in females [83, 84]. Men have increased gonadotropins as they age, but this is not necessarily associated with changes in T [85] or dementia [84]. Notably, androgen deprivation therapy in men with GnRH agonists ± antiandrogens (low gonadotropin conditions) and castration (high gonadotropin conditions) are both associated with increased stroke risk, whereas antiandrogen monotherapy (high gonadotropin conditions) is not [38, 86]. Similarly, in a study comparing GnRH agonist to castration in prostate cancer patients showed similar levels of cardiovascular ischemic events, despite one condition being a high gonadotropin and one being a low gonadotropin condition [87]. Future studies to specifically examine the role of gonadotropins or other testicular factors on cognition will be needed to address these apparent inconsistencies. Regardless of mechanism, the results of the present study support clinical findings that show modest cognitive benefits of TRT in men under 60 [88, 89], but not older cohorts [90, 91].

Androgens can have actions on many cells in the brain, but the sites of detrimental effects after ischemia are not known. One possibility comes from male mice in which 9 weeks of hypogonadism reduces the integrity of the BBB and endothelial tight junctions and increases the inflammatory response of astrocytes and microglia [92]. Castration similarly enhances oxidative stress leading to endothelial and neuronal senescence in senescence accelerated mice, in part through enhancing vascular inflammation [93]. T reverses this effect and enhances endothelial nitric oxide production [93]. Interestingly, all of the castrate groups in the present study showed enhanced vasoconstrictor responses to ET-1, even in the context of TRT. Such a result suggests that cerebrovascular responses to castration may be refractory to T, since several studies support a vasorelaxant effect of T [94]. Nevertheless, the detrimental role of T in the long-term castrate group is directly comparable to the long-term castrate controls with cholesterol implants. Both groups were castrate and had a similar fall in cerebral perfusion, but different behavioral outcomes.

Several mechanisms have been proposed to account for the loss of estrogen-dependent neuroprotection following long-term ovariectomy, including changes in receptor expression, micro RNAs, inflammation, and vascular reactivity [57]. Similar mechanisms may be in play for the detrimental effect of TRT observed in the present study. We chose to treat with T rather than a non-aromatizable androgen because testosterone is used clinically in the treatment of hypogonadism. However, both the protective effects on cognition and the detrimental effects in LTTD rats after stroke may be dependent on the conversion of T to estrogen in the brain. Cheng et al. [32] observed increased aromatase activity in the striatum of young, but not middle-aged rats following experimental stroke, but aromatase knockout mice had similar injury as wild-type animals. Furthermore, the androgen receptor antagonist flutamide blocked the detrimental effects of T in young animals and the beneficial effects in middle-aged animals, suggesting that conversion to estrogen was not responsible for the effects of T [32]. Nevertheless, future studies are needed to determine which steroid metabolites and receptors are responsible for the detrimental effects observed after LTTD.

The use of TRT, especially in young men (<50), without diagnosed organic hypogonadism remains controversial, and it is likely that the balance of effects of TRT will be dependent on comorbidities. Results of the present study suggest that factors that lead to increased oxidative stress, including long-term hypogonadism, may predispose the ischemic brain to exaggerated injury. Indeed, even in the uninjured state, peripheral oxidative stress was highly correlated with poor cognitive performance, similar to the effects observed in men with low T [95]. Although the castrate rat model may not fully reflect men with idiopathic low T, regardless of the reason for hypogonadism, our results support early rather than delayed TRT. Future studies will be required to assess which comorbidities put men at high risk for adverse outcomes and whether the beneficial effects of TRT on cognition, libido, and mood outweigh potential risks.

The authors wish to acknowledge the assistance of E. Michael Cueller and Callie Fort in blood and tissue processing and behavioral analysis.

The authors have no conflicts of interest to declare.

This project was funded by NIH 5RO3AG049255 (D.A.S. and R.L.C.) and a seed grant from the University of North Texas Health Science Center (D.A.S. and R.L.C.).

C.S.: animal studies including treatments, surgery, stroke and behavioral testing, data analysis, and manuscript preparation. J.C.-G.: animal cognition studies, surgery, and data analysis. R.L.C. and D.A.S.: overall design, direction, and manuscript preparation. J.M.W. and P.H.V.: animal behavior testing. D.M.: animal care. E.K. and A.O.-G.: ELISAs. N.S.: direction of behavioral studies and analysis of behavioral data, and manuscript preparation.

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