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Intranasal administration of testosterone increased immobile-sniffing, exploratory behavior, motor behavior and grooming behavior in rats
Hormones and Behavior 59 (2011) 477–483
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Hormones and Behavior j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y h b e h
Intranasal administration of testosterone increased immobile-sniffing, exploratory behavior, motor behavior and grooming behavior in rats Guoliang Zhang a, Geming Shi a,⁎, Huibing Tan b, Yunxiao Kang a, Huixian Cui c a b c
Department of Neurobiology, Hebei Medical University, Shijiazhuang, PR China Department of Anatomy and Cell Biology, The Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada Department of Human Anatomy, Hebei Medical University, Shijiazhuang, PR China
a r t i c l e
i n f o
Article history: Received 10 October 2010 Revised 19 January 2011 Accepted 21 January 2011 Available online 31 January 2011 Keywords: Intranasal administration Testosterone Open field Behavior Rats
a b s t r a c t Currently, testosterone (T) replacement therapy is typically provided by oral medication, injectable T esters, surgically implanted T pellets, transdermal patches and gels. However, most of these methods of administration are still not ideal for targeting the central nervous system. Recently, therapeutic intranasal T administration (InT) has been considered as another option for delivering T to the brain. In the present study, the effects of 21-day InT treatment were assessed on open field behavior in gonadectomized (GDX) rats and intact rats. Subcutaneous injections of T at same dose were also tested in GDX rats. A total of 12 behavioral events were examined in GDX groups with or without T and in intact groups with or without InT. Significant decreases in open field activity were observed in rats after GDX without InT compared to sham-operated rats. The open field activity scores for most tests significantly increased with InT treatment in GDX rats and in intact rats compared with the corresponding GDX rats and intact rats. Intranasal administration of T improved the reduced behaviors resulted from T deficiency better than subcutaneous injection of T, demonstrating that T can be delivered to the brain by intranasal administration. Our results suggest that intranasal T delivery is an effective option for targeting the central nervous system. © 2011 Elsevier Inc. All rights reserved.
Introduction Numerous studies have demonstrated that anabolic androgenic steroids (AASs), which include the endogenous male hormone testosterone (T) and structurally related synthetic compounds (Hoberman and Yesalis, 1995), can influence behaviors (Frye and Seliga, 2001; Lambadjieva, 1999; Perry et al., 2003). Subcutaneous administration of T enhances anti-anxiety behavior in the elevated plus maze, the zero maze and the Vogel task and also increases motor behavior in the activity monitoring test in aged intact male C57/B6 mice (Frye et al., 2008). Gonadectomy decreases open field activity in male rats and supplementation with testosterone propionate in GDX rats recovers open field activity (Adler et al., 1999). Intact or GDX rats that were treated intramuscularly with nandrolone decanoate spend more time in the margin of the open-field (Minkin et al., 1993). Androgen-treated rats (subcutaneous or intrahippocampal) showed significantly more exploratory behavior in the open field (Edinger and Frye, 2005). At present, testosterone replacement includes subcutaneous T implants, intramuscular injections and oral therapy (Gold and Voskuhl, 2006; Handelsman et al., 1997; Jordan, 1997; Nieschlag, 2006; Parker and Armitage, 1999). The intranasal route offers the possibility of ⁎ Corresponding author at: Department of Neurobiology, Hebei Medical University, Post code 050017, Shijiazhuang, PR China. E-mail address: [email protected] (G. Shi). 0018-506X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2011.01.007
bypassing the first-pass metabolism and potentially provides a direct delivery of the drug to the central nervous system (Banks et al., 2009; Costantino et al., 2007; de Souza Silva et al., 2009; Hanson and Frey, 2008; Illum, 2007; Pires et al., 2009). Intranasal delivery of T might be an alternative method of administration (Banks et al., 2009; de Souza Silva et al., 2009; Mattern et al., 2008). Therefore, the purpose of the present paper was to study the effects of intranasal administration of T on the central nervous system by analyzing the open field behavior of rats after long-term intranasal administration of T.
Materials and methods Animals and housing Sixty-four adult male Wistar rats (252 ± 5 g) were supplied by the Experimental Animal Center of Hebei Medical University. Before treatment, the animals were fed in the experimental room for 3 days to acclimate to the environment of the laboratory. All of the animals were kept in standard Plexiglas cages in groups of four per cage. The animals were housed under controlled temperature (22 °C) and humidity conditions with a 12-h light–dark cycle (lights on 06:00 h). Food and water were available ad libitum. All of the experimental procedures followed the rules in the “Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research” and were
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approved by the Committee of Ethics on Animal Experiments at Hebei Medical University. Surgery For intranasal administration experiment, 40 rats were randomly assigned to one of five groups: intact (n = 8) and intact rats that were intranasally supplemented with T (intact-T) (n = 8), sham-operated (sham) (n = 8), gonadectomized (GDX) (n = 8), GDX rats that were intranasally supplemented with T (GDX-T) (n = 8). For subcutaneous injection experiment, 24 rats were randomly assigned to one of three groups: sham-operated (sham-sc) (n = 8), gonadectomized (GDX-sc) (n = 8), GDX rats that were subcutaneously supplemented with T (GDX-sc.T) (n = 8). Animals were anesthetized with an intraperitoneal injection (i.p.) of chloral hydrate (300 mg/kg). All of the surgeries were carried out under aseptic conditions. The GDX, GDX-T, GDX-sc and GDX-sc.T rats received bilateral orchiectomies (ORXs) that involved the removal of the testes, epididymis, and epididymal fat. The incisions were closed using surgical staples. The sham-operated rats experienced the same surgical treatment except for the ORXs. Drugs and treatments Testosterone (product code: 1616690, International Laboratory, USA) was dissolved in sesame oil and then intranasally administered to GDX-T rats and intact-T rats or subcutaneously to GDX-sc.T rats at a dose of 2.0 mg/kg, at 5:00 PM to 6:00 PM, once a day. Intranasal administration was performed as described (Rojo et al., 2006). Rats were held and laid upside down. Testosterone solution was introduced by the pressure with a micropipette into one nasal cavity (25 μl), without introducing the pipette tip directly into the nasal cavity. Afterwards, the rats were immobilized in this position for 15 s by gently pulling the tail. After 3 min the same procedure was repeated in the other nostril. This procedure was repeated daily for 21 days. The GDX rats, sham rats and intact rats received the same treatment using sesame oil as a vehicle. The sham-sc rats and GDX-sc rats did the subcutaneous treatment using sesame oil as a vehicle. The open field Apparatus The open field apparatus was a box (100 × 100 × 40 cm) constructed from plastic board that consisted of four black walls and a
white bottom without a cover. The bottom was lined into 25 squares (20 × 20 cm). Every square further consisted of 400 small grills (1 × 1 cm). The apparatus was located in a sound-attenuating chamber and was illuminated with a luminance of 20 lux on the floor. A digital video camera (Canon HF100, Japan) was installed above the apparatus. To neutralize odors, the arena was cleaned with 70% ethanol before each subject was tested. Procedure All of the animals were tail-marked and handled for 5 days prior to the test. Experiments were performed during the animals’ inactive period between 8:00 AM and 6:00 PM. On the 18th day, the rats were pre-exposed to the open field apparatus for 5 min. On the 19th day, the rats were pre-exposed to the open field apparatus for 15 min. On the 20th and the 21st day, each rat was individually placed in the center of the open field apparatus and allowed to explore the field for 15 min. The rats' performances on the 20th and the 21st days were recorded and analyzed post hoc. Parameters For the analysis, the field was divided into center squares and peripheral squares. Five types of behavioral patterns were analyzed in our study: immobile-sniffing, exploratory behavior, thigmotaxic behavior, motor behavior and grooming behavior (Table 1). The 2-day continuous behavior parameters were scored by the observers blind to the experimental plan and registered by shorthand. We observed that the data was consistent for both days. Since there were no substantial differences in most behavior parameters on the 20th day and the 21st day (ANOVA), the results are averaged for each rat. The averaged amount of individual behavior parameters was presented for each rat in the results. Statistical analysis All of the behavioral data are presented as the mean ± SD. We applied the tests of normality (Kolmogorov–Smirnov test) and homogeneity variance (Levene's test) to all behavioral data. If both normal distribution (P N 0.1) and homogeneity of variance (P N 0.1) were found, then parametric test was performed by one-way analysis of variance (one-way ANOVA) followed by a Student–Newman–Keuls (SNK) post hoc test for multiple comparisons. Otherwise, we used non-parametric statistics by a Kruskal–Wallis test; where P b 0.05, post-hoc between group were done using the Mann–Whitney U test.
Table 1 Behavior patterns of rat in the open field test. Behavior pattern
Unit of parameters The action of rat
Immobile-sniffing Exploratory behavior Walking Climbing Rearing Sniffing
number
Rat sniffs the environment standing on the ground (Casarrubea et al., 2008, 2009a, 2009b)
number number number number
Rat walks around sniffing the environment (Casarrubea et al., 2008, 2009a, 2009b) Rat maintains an erect posture leaning against the wall (Casarrubea et al., 2008, 2009a, 2009b) Rat maintains an erect posture without leaning against the wall (Casarrubea et al., 2008, 2009a, 2009b) Rat sniffs the environment in moving (Walking + Climbing + Rearing) (Casarrubea et al., 2008, 2009a, 2009b; Meyerson and Höglund, 1981) Rat prefers the periphery of the apparatus to activity in the central parts of the open field (Prut and Belzung, 2003) Total time spent in the peripheral squares in whole test period
Thigmotaxic behavior Time spent in the peripheral squares second Motor behavior Vertical activity number Horizontal activity number Total path length Grooming behavior Latency of grooming Number of grooming Duration of grooming
centimeter
second number number
Total number of erect posture in whole test period (Climbing+ Rearing) (Ericson et al., 1991; Prut and Belzung, 2003) Total number of square crossings in whole test period (3 or more paws moved from the original square to an adjacent one) (Ericson et al., 1991; Hillegaart et al., 1989; Prut and Belzung, 2003; Wultz et al., 1990) Total length of crossings in whole test period (Li and Huang, 2006) Include rat paw licking, nose/face grooming, head washing, body grooming/scratching, leg licking and tail/genitals grooming (Kalueff and Tuohimaa, 2004, 2005; Kalueff et al., 2007) Time from the onset of the test until the grooming behavior was first displayed Number of all kind of grooming in whole test period Total time of grooming behavior in whole test period
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procedures and vehicle delivery had no effect on measures of behaviors. The effect of intranasal administration of testosterone on open field behavior
Fig. 1. Effects of intranasal administration and subcutaneous injection of T on the immobile-sniffing. The asterisks show significant differences (*P b 0.01).
Differences were considered to be significant when P values were less than 0.05. Results No significant differences on behaviors were observed among sham, sham-sc and intact rats, demonstrating that sham-operated
Immobile-sniffing We first examined the immobile-sniffing of rats. The intranasal administration of T in intact rats significantly increased the number of immobile-sniffing events by 46% (Fig. 1; one-way ANOVA, F(1,14) = 11.032, P b 0.01). Group differences in the number of immobilesniffing events were found among the sham, GDX and GDX-T rats (Fig. 1; Kruskal–Wallis test, χ2 = 17.740, P b 0.01). The post hoc test showed that gonadectomy decreased the immobile-sniffing events, with a 44% (P b 0.01) reduction and the intranasal administration of T in GDX rats significantly increased the number of immobile-sniffing events (P b 0.01), which was even more than in sham rats by 88% (P b 0.01). Exploratory behaviors We next observed the exploratory behaviors of rats. The intranasal administration of T significantly increased the amount of walking, climbing, rearing and sniffing in intact rats by 82% (Fig. 2A; Kruskal– Wallis test, χ2 = 7.049, P b 0.01), 54% (Fig. 2B; one-way ANOVA, F(1,14) = 8.327, P b 0.05), 85% (Fig. 2C; Kruskal–Wallis test, χ2 = 7.236, P b 0.01) and 65% (Fig. 2D; Kruskal–Wallis test, χ2 = 7.757, P b 0.01), respectively. Group differences among the sham, GDX and GDX-T rats
Fig. 2. Effects of intranasal administration and subcutaneous injection of T on the amount of walking (A), climbing (B), rearing (C) and sniffing (D). The asterisks indicate significant differences (*P b 0.05, **P b 0.01).
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were found in the amount of walking (Kruskal–Wallis test, χ2 = 16.107, P b 0.01), climbing (one-way ANOVA, F(2,21) = 12.129, P b 0.01), rearing (Kruskal–Wallis test, χ2 = 13.528, P b 0.01) and sniffing (Kruskal–Wallis test, χ2 = 14.495, P b 0.01). The post hoc test indicated that the amount of walking, climbing, rearing and sniffing in GDX rats was significantly lower than in the sham rats by 70% (Fig. 2A; P b 0.01), 57% (Fig. 2B; P b 0.01), 68% (Fig. 2C; P b 0.01) and 62% (Fig. 2D; P b 0.01), respectively, and that the amount of above behaviors in GDX rats was restored to the level of sham rats after intranasal administration of T, except for the rearing. The amount of rearing in GDX-T rats was still lower than in the sham rats by 47% (P b 0.05), even though the amount was increased 68% (Fig. 2C; P N 0.05) compared to GDX rats. Thigmotaxic behavior The effect of intranasal administration of T was further tested in terms of the thigmotaxic behavior of rats. No differences were observed for thigmotaxic behavior (data not shown). Motor behaviors Vertical activity, horizontal activity and total path length were then examined in the motor behavior experiments. The intranasal administration of T significantly increased the amount of vertical activity and horizontal activity as well as the total path length in intact rats by 61% (Fig. 3A; one-way ANOVA, F(1,14) = 13.002, P b 0.01), 69% (Fig. 3B; Kruskal–Wallis test, χ2 = 6.353, P b 0.05) and 75% (Fig. 3C; one-way ANOVA, F(1,14) = 10.884, P b 0.01), respectively. Group differences among the sham, GDX and GDX-T rats were found in the
amount of vertical activity (one-way ANOVA, F(2,21) = 16.485, P b 0.01), horizontal activity (Kruskal–Wallis test, χ2 = 12.214, P b 0.01) and in the total path length (one-way ANOVA, F(2,21)= 14.446, P b 0.01). The post hoc test found that the amount of vertical activity and horizontal activity in GDX rats was significantly lower than in the sham rats by 60% (Fig. 3A; P b 0.01) and 60% (Fig. 3B; P b 0.01), respectively. The total path length in GDX rats was significantly less than in the sham rats by 59% (Fig. 3C; P b 0.01) and the amount of vertical activity and horizontal activity as well as the total path length in GDX rats were restored to the level of sham rats after intranasal administration of T.
Grooming behaviors Finally, grooming behaviors were tested. No differences were observed for the latency of grooming (data not shown). The intranasal administration of T significantly increased the number of grooming events and the duration of grooming in intact rats by 72% (Fig. 4A; one-way ANOVA, F(1,14) = 13.138, P b 0.01) and 61% (Fig. 4B; oneway ANOVA, F(1,14) = 8.755, P b 0.01), respectively. Group differences among the sham, GDX and GDX-T rats were found in the number of grooming events (Kruskal–Wallis test, χ2 = 13.598, P b 0.01) and the duration of grooming (one-way ANOVA, F(2,21) = 8.658, P b 0.01). The post hoc test revealed that the number of grooming events in GDX rats was significantly less than in the sham rats by 54% (Fig. 4A; P b 0.01), the duration of grooming in GDX rats was significantly shorter than in the sham rats by 55% (Fig. 4B; P b 0.01) and the number of grooming events as well as the duration of
Fig. 3. Effects of intranasal administration and subcutaneous injection of T on the amount of vertical activity (A) and horizontal activity (B) and total path length (C). The asterisks mark significant differences (*P b 0.05, **P b 0.01).
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Fig. 4. Effects of intranasal administration and subcutaneous injection of T on the number of grooming events (A) and the duration of grooming (B). The asterisks indicate significant differences (*P b 0.01).
grooming in GDX rats was restored to the level of sham rats after intranasal administration of T. The effect of subcutaneous injection of testosterone on open field behavior Significant decreases in open field activity were observed in GDXsc rats (Figs. 1–4). The subcutaneous injection of T only restored the amount of walking (Fig. 2A), horizontal activity (Fig. 3B) and the total path length (Fig. 3C) to the level of sham-sc rats. There were no significant differences between GDX-sc.T and GDX-sc rats in the number of immobile-sniffing events, the amount of climbing, rearing, vertical activity, the number of grooming events and the duration of grooming, except for the amount of sniffing (Fig. 2D). In total, we observed 12 kinds of behavioral operations in the open field test (Table 1). The open field activity scores for most tests significantly increased with intranasal delivery of T in GDX rats and intact rats. Intranasal administration of T could restore the most of parameters of open field behavior in GDX rats, however, only a few behavioral operations were improved in GDX-sc rats after subcutaneous T administration treatment. Discussion In the present study, we found that the parameters of immobilesniffing, exploratory behavior, motor behavior and grooming behavior in rats were significantly reduced after the rats were gonadectomized.
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Intranasal administration of T to GDX rats and intact rats increased the above behaviors. The most of parameters of open field behavior were improved in GDX-T rats. Our results indicated that chronic nasal administration of T could affect the central nervous system of rats. It was found that the T levels in the blood after nasal and intravenous administration were similar in rats and the bioavailability of the nasally administered T was over 90% (Hussain et al., 1984). In mice, the bioavailability in serum was found to be 75% after intranasal administration (Banks et al., 2009). Testosterone enters the brain through direct and indirect transport routes after intranasal administration. About two thirds of T in the brain after intranasal administration directly entered the brain from the nasal area without first being absorbed into the blood and the remainder indirectly entered the brain by first entering the blood and then crossing the blood–brain barrier (BBB) (Banks et al., 2009). In our study, the intranasal administration of T could improve the most of the reduced parameters of open field behavior in GDX rats, but, only a few behavioral parameters were improved in GDX-sc rats when T was given subcutaneously at same dose. These results in the present experiments suggested that intranasal administration of T is a more efficacious way of targeting the brain, which was inferred from a recent study that intranasal administration of T at the dose of 2.0 mg/ kg could effectively increase dopaminergic activity in the neostriatum and nucleus accumbens, however, subcutaneous administration of T at the same dose did not lead to an increase in dopamine in the neostriatum and nucleus accumbens and the increased dopamine was only seen at higher doses of 8.0 mg/kg in the neostriatum (de Souza Silva et al., 2009). Intranasal administration of T produced a targeted delivery to the brain, especially to the olfactory bulbs, hypothalamus, striatum, and hippocampus (Banks et al., 2009). The difference of regional brain distribution of T after intranasal administration might be related to the regional differences in androgen receptor binding. Androgen receptor mRNA-containing neurons were heavily distributed in the olfactory bulbs, hypothalamus and hippocampus (Simerly et al., 1990). The difference in terms of the effect potency of the intranasal delivery of T on the behaviors of rats between GDX-T vs. GDX and intact-T vs. intact in our study is probably due to the different serum T levels in GDX (the absence of endogeneous gonadal hormones) and intact (the presence of endogeneous gonadal hormones) rats. There are two factors that should be further investigated in the future studies on the effects of the intranasal administration of T on the open field behavior. One is T dose delivered intranasally to GDX rats. The dose in the present study was chosen on the basis of the findings that the intranasal application of 2.0 mg/kg of T resulted in an increase in dopamine levels in both the neostriatum and nucleus accumbens (de Souza Silva et al., 2009). Intranasal delivery of 2.0 mg/ kg/day of T to GDX rats improved the most of parameters of open field behavior but did not fully restore the reduced behaviors in GDX rats. Different doses should be further tested in the following study. Another is sex hormone-binding globulin, which limits T ability to enter the brain from blood stream and is absent in rodent animals (Downer et al., 2001). Studies of the effect of intranasal delivery of T on the open field behavior should be further performed in non-rodent species. Testosterone can easily cross BBB due to its lipophilic properties, but systemic administration of T has been shown to be problematic. The efficacy of oral testosterone undecanoate is limited because of its unreliable oral bioavailability, fluctuating serum levels and short halflife necessitating multiple daily doses. Testosterone taken orally is also rapidly inactivated by first-pass hepatic metabolism, which makes oral therapy an ineffective means of delivering T. Deep intramuscular injections are invasive and can cause patient discomfort (Nieschlag et al., 2004). The insertion of T implants requires minor surgery and can be painful, and extrusion of the pellets is common (Handelsman et al., 1997). Transdermal T non-scrotal patches result
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in skin irritation in patients (Parker and Armitage, 1999; Jordan, 1997). Testosterone gels are not always considered convenient and bear the risk of skin-to-skin transfer (Rolf et al., 2002). The ideal T replacement therapy should offer safety, efficacy, cost-effectiveness, convenience, a good release profile, dosing flexibility, and effective normalization of T levels (Nieschlag et al., 2004). Targeting T to the central nervous system should allow the administration of lower doses of T and fewer peripheral side effects. The intranasal route would seem to be advantageous for T administration. Lipophilic substances, such as T, are transported directly from the nose to the brain through: (1) the olfactory epithelial pathways (across the sustentacular cells, through tight junctions between sustentacular cells, or clefts between sustentacular cells and olfactory neurons), (2) the olfactory nerve pathway, or (3) through the trigeminal nerve pathway (Graff and Pollack, 2005; Illum, 2004, 2007; Thorne et al., 2004). This delivery route would also negate the effects of serum protein binding so that the intranasal route dose of T needed for brain delivery would likely be lower than the systemic dose (Morley et al., 2002). Testosterone was administered intranasally in anesthetized male rats, and its effects on the activity of dopaminergic and serotonergic neurons in the neostriatum and nucleus accumbens were assessed by means of microdialysis and high performance liquid chromatography (HPLC). The intranasal administration of T led to an immediate increase in DA and 5-HT levels in both the neostriatum and nucleus accumbens (de Souza Silva et al., 2009), which indicated that intranasal administration of T rapidly activated central dopaminergic and serotonergic systems. Increased behavioral scores in the open field test after chronic intranasal delivery of T might result from the altered dopaminergic and serotonergic system. Immunocytochemical (DonCarlos et al., 1991; Kritzer, 1997) and in situ hybridization (Simerly et al., 1990) studies identified subpopulations of intracellular gonadal hormone receptor-bearing neurons in the substantia nigra (SN) and the ventral tegmental area (VTA), which suggest that specific subsets of midbrain neurons might be direct targets of gonadal hormones. It was found that nearly every androgen receptor bearing cell in the VTA and SN pars compacta, roughly half in the SN pars lateralis, and about one-third in the retrorubral fields were tyrosine hydroxylase immunopositive and these androgen receptors tend to occupy regions labeled by injections in limbic or cortical targets (Kritzer, 1997; Creutz and Kritzer, 2004). Chronic intranasal administration of T might result in transcriptional changes in protein synthesis via intracellular androgen receptors in the mesolimbic and mesocortical DA neurons and influence behavioral functions. Lower androgenic level or T deficit influenced organism. Testosterone deficiency disorder could refer to premature mortality and to a number of co-morbidities, such as sexual disorders and diabetes as well as metabolic syndrome. Testosterone deficiency occurs mainly in ageing men when prostate disease starts to emerge. The current T supplements by different route of administration developed during the last decade of these patients (Raynaud, 2009). When intranasal administration of T is also expected to be used as adjunctive therapy for some neuropsychiatric disorders, such as PD (Mitchell et al., 2006), the direct nasal route of T delivery to brain would become the dominant factor in determining brain uptake because the ability of T to enter the brain is limited in the presence of sex hormone-binding globulin in serum (Downer et al., 2001; Hobbs et al. 1992; Pardridge et al. 1980). Gonadectomy in male rats decreased immobile-sniffing, exploratory behavior, motor behavior and grooming behavior. There is not a single supplement that can treat it all. The impaired behaviors insulted by gonadectomy could be mostly restored by chronic intranasal administration of T, which is better than subcutaneous injection of T, demonstrating that T can be delivered to the brain to affect the brain functions by intranasal administration. Therefore, the intranasal T delivery is an effective option for targeting the central
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Hormones and Behavior j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y h b e h
Intranasal administration of testosterone increased immobile-sniffing, exploratory behavior, motor behavior and grooming behavior in rats Guoliang Zhang a, Geming Shi a,⁎, Huibing Tan b, Yunxiao Kang a, Huixian Cui c a b c
Department of Neurobiology, Hebei Medical University, Shijiazhuang, PR China Department of Anatomy and Cell Biology, The Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada Department of Human Anatomy, Hebei Medical University, Shijiazhuang, PR China
a r t i c l e
i n f o
Article history: Received 10 October 2010 Revised 19 January 2011 Accepted 21 January 2011 Available online 31 January 2011 Keywords: Intranasal administration Testosterone Open field Behavior Rats
a b s t r a c t Currently, testosterone (T) replacement therapy is typically provided by oral medication, injectable T esters, surgically implanted T pellets, transdermal patches and gels. However, most of these methods of administration are still not ideal for targeting the central nervous system. Recently, therapeutic intranasal T administration (InT) has been considered as another option for delivering T to the brain. In the present study, the effects of 21-day InT treatment were assessed on open field behavior in gonadectomized (GDX) rats and intact rats. Subcutaneous injections of T at same dose were also tested in GDX rats. A total of 12 behavioral events were examined in GDX groups with or without T and in intact groups with or without InT. Significant decreases in open field activity were observed in rats after GDX without InT compared to sham-operated rats. The open field activity scores for most tests significantly increased with InT treatment in GDX rats and in intact rats compared with the corresponding GDX rats and intact rats. Intranasal administration of T improved the reduced behaviors resulted from T deficiency better than subcutaneous injection of T, demonstrating that T can be delivered to the brain by intranasal administration. Our results suggest that intranasal T delivery is an effective option for targeting the central nervous system. © 2011 Elsevier Inc. All rights reserved.
Introduction Numerous studies have demonstrated that anabolic androgenic steroids (AASs), which include the endogenous male hormone testosterone (T) and structurally related synthetic compounds (Hoberman and Yesalis, 1995), can influence behaviors (Frye and Seliga, 2001; Lambadjieva, 1999; Perry et al., 2003). Subcutaneous administration of T enhances anti-anxiety behavior in the elevated plus maze, the zero maze and the Vogel task and also increases motor behavior in the activity monitoring test in aged intact male C57/B6 mice (Frye et al., 2008). Gonadectomy decreases open field activity in male rats and supplementation with testosterone propionate in GDX rats recovers open field activity (Adler et al., 1999). Intact or GDX rats that were treated intramuscularly with nandrolone decanoate spend more time in the margin of the open-field (Minkin et al., 1993). Androgen-treated rats (subcutaneous or intrahippocampal) showed significantly more exploratory behavior in the open field (Edinger and Frye, 2005). At present, testosterone replacement includes subcutaneous T implants, intramuscular injections and oral therapy (Gold and Voskuhl, 2006; Handelsman et al., 1997; Jordan, 1997; Nieschlag, 2006; Parker and Armitage, 1999). The intranasal route offers the possibility of ⁎ Corresponding author at: Department of Neurobiology, Hebei Medical University, Post code 050017, Shijiazhuang, PR China. E-mail address: [email protected] (G. Shi). 0018-506X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2011.01.007
bypassing the first-pass metabolism and potentially provides a direct delivery of the drug to the central nervous system (Banks et al., 2009; Costantino et al., 2007; de Souza Silva et al., 2009; Hanson and Frey, 2008; Illum, 2007; Pires et al., 2009). Intranasal delivery of T might be an alternative method of administration (Banks et al., 2009; de Souza Silva et al., 2009; Mattern et al., 2008). Therefore, the purpose of the present paper was to study the effects of intranasal administration of T on the central nervous system by analyzing the open field behavior of rats after long-term intranasal administration of T.
Materials and methods Animals and housing Sixty-four adult male Wistar rats (252 ± 5 g) were supplied by the Experimental Animal Center of Hebei Medical University. Before treatment, the animals were fed in the experimental room for 3 days to acclimate to the environment of the laboratory. All of the animals were kept in standard Plexiglas cages in groups of four per cage. The animals were housed under controlled temperature (22 °C) and humidity conditions with a 12-h light–dark cycle (lights on 06:00 h). Food and water were available ad libitum. All of the experimental procedures followed the rules in the “Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research” and were
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approved by the Committee of Ethics on Animal Experiments at Hebei Medical University. Surgery For intranasal administration experiment, 40 rats were randomly assigned to one of five groups: intact (n = 8) and intact rats that were intranasally supplemented with T (intact-T) (n = 8), sham-operated (sham) (n = 8), gonadectomized (GDX) (n = 8), GDX rats that were intranasally supplemented with T (GDX-T) (n = 8). For subcutaneous injection experiment, 24 rats were randomly assigned to one of three groups: sham-operated (sham-sc) (n = 8), gonadectomized (GDX-sc) (n = 8), GDX rats that were subcutaneously supplemented with T (GDX-sc.T) (n = 8). Animals were anesthetized with an intraperitoneal injection (i.p.) of chloral hydrate (300 mg/kg). All of the surgeries were carried out under aseptic conditions. The GDX, GDX-T, GDX-sc and GDX-sc.T rats received bilateral orchiectomies (ORXs) that involved the removal of the testes, epididymis, and epididymal fat. The incisions were closed using surgical staples. The sham-operated rats experienced the same surgical treatment except for the ORXs. Drugs and treatments Testosterone (product code: 1616690, International Laboratory, USA) was dissolved in sesame oil and then intranasally administered to GDX-T rats and intact-T rats or subcutaneously to GDX-sc.T rats at a dose of 2.0 mg/kg, at 5:00 PM to 6:00 PM, once a day. Intranasal administration was performed as described (Rojo et al., 2006). Rats were held and laid upside down. Testosterone solution was introduced by the pressure with a micropipette into one nasal cavity (25 μl), without introducing the pipette tip directly into the nasal cavity. Afterwards, the rats were immobilized in this position for 15 s by gently pulling the tail. After 3 min the same procedure was repeated in the other nostril. This procedure was repeated daily for 21 days. The GDX rats, sham rats and intact rats received the same treatment using sesame oil as a vehicle. The sham-sc rats and GDX-sc rats did the subcutaneous treatment using sesame oil as a vehicle. The open field Apparatus The open field apparatus was a box (100 × 100 × 40 cm) constructed from plastic board that consisted of four black walls and a
white bottom without a cover. The bottom was lined into 25 squares (20 × 20 cm). Every square further consisted of 400 small grills (1 × 1 cm). The apparatus was located in a sound-attenuating chamber and was illuminated with a luminance of 20 lux on the floor. A digital video camera (Canon HF100, Japan) was installed above the apparatus. To neutralize odors, the arena was cleaned with 70% ethanol before each subject was tested. Procedure All of the animals were tail-marked and handled for 5 days prior to the test. Experiments were performed during the animals’ inactive period between 8:00 AM and 6:00 PM. On the 18th day, the rats were pre-exposed to the open field apparatus for 5 min. On the 19th day, the rats were pre-exposed to the open field apparatus for 15 min. On the 20th and the 21st day, each rat was individually placed in the center of the open field apparatus and allowed to explore the field for 15 min. The rats' performances on the 20th and the 21st days were recorded and analyzed post hoc. Parameters For the analysis, the field was divided into center squares and peripheral squares. Five types of behavioral patterns were analyzed in our study: immobile-sniffing, exploratory behavior, thigmotaxic behavior, motor behavior and grooming behavior (Table 1). The 2-day continuous behavior parameters were scored by the observers blind to the experimental plan and registered by shorthand. We observed that the data was consistent for both days. Since there were no substantial differences in most behavior parameters on the 20th day and the 21st day (ANOVA), the results are averaged for each rat. The averaged amount of individual behavior parameters was presented for each rat in the results. Statistical analysis All of the behavioral data are presented as the mean ± SD. We applied the tests of normality (Kolmogorov–Smirnov test) and homogeneity variance (Levene's test) to all behavioral data. If both normal distribution (P N 0.1) and homogeneity of variance (P N 0.1) were found, then parametric test was performed by one-way analysis of variance (one-way ANOVA) followed by a Student–Newman–Keuls (SNK) post hoc test for multiple comparisons. Otherwise, we used non-parametric statistics by a Kruskal–Wallis test; where P b 0.05, post-hoc between group were done using the Mann–Whitney U test.
Table 1 Behavior patterns of rat in the open field test. Behavior pattern
Unit of parameters The action of rat
Immobile-sniffing Exploratory behavior Walking Climbing Rearing Sniffing
number
Rat sniffs the environment standing on the ground (Casarrubea et al., 2008, 2009a, 2009b)
number number number number
Rat walks around sniffing the environment (Casarrubea et al., 2008, 2009a, 2009b) Rat maintains an erect posture leaning against the wall (Casarrubea et al., 2008, 2009a, 2009b) Rat maintains an erect posture without leaning against the wall (Casarrubea et al., 2008, 2009a, 2009b) Rat sniffs the environment in moving (Walking + Climbing + Rearing) (Casarrubea et al., 2008, 2009a, 2009b; Meyerson and Höglund, 1981) Rat prefers the periphery of the apparatus to activity in the central parts of the open field (Prut and Belzung, 2003) Total time spent in the peripheral squares in whole test period
Thigmotaxic behavior Time spent in the peripheral squares second Motor behavior Vertical activity number Horizontal activity number Total path length Grooming behavior Latency of grooming Number of grooming Duration of grooming
centimeter
second number number
Total number of erect posture in whole test period (Climbing+ Rearing) (Ericson et al., 1991; Prut and Belzung, 2003) Total number of square crossings in whole test period (3 or more paws moved from the original square to an adjacent one) (Ericson et al., 1991; Hillegaart et al., 1989; Prut and Belzung, 2003; Wultz et al., 1990) Total length of crossings in whole test period (Li and Huang, 2006) Include rat paw licking, nose/face grooming, head washing, body grooming/scratching, leg licking and tail/genitals grooming (Kalueff and Tuohimaa, 2004, 2005; Kalueff et al., 2007) Time from the onset of the test until the grooming behavior was first displayed Number of all kind of grooming in whole test period Total time of grooming behavior in whole test period
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procedures and vehicle delivery had no effect on measures of behaviors. The effect of intranasal administration of testosterone on open field behavior
Fig. 1. Effects of intranasal administration and subcutaneous injection of T on the immobile-sniffing. The asterisks show significant differences (*P b 0.01).
Differences were considered to be significant when P values were less than 0.05. Results No significant differences on behaviors were observed among sham, sham-sc and intact rats, demonstrating that sham-operated
Immobile-sniffing We first examined the immobile-sniffing of rats. The intranasal administration of T in intact rats significantly increased the number of immobile-sniffing events by 46% (Fig. 1; one-way ANOVA, F(1,14) = 11.032, P b 0.01). Group differences in the number of immobilesniffing events were found among the sham, GDX and GDX-T rats (Fig. 1; Kruskal–Wallis test, χ2 = 17.740, P b 0.01). The post hoc test showed that gonadectomy decreased the immobile-sniffing events, with a 44% (P b 0.01) reduction and the intranasal administration of T in GDX rats significantly increased the number of immobile-sniffing events (P b 0.01), which was even more than in sham rats by 88% (P b 0.01). Exploratory behaviors We next observed the exploratory behaviors of rats. The intranasal administration of T significantly increased the amount of walking, climbing, rearing and sniffing in intact rats by 82% (Fig. 2A; Kruskal– Wallis test, χ2 = 7.049, P b 0.01), 54% (Fig. 2B; one-way ANOVA, F(1,14) = 8.327, P b 0.05), 85% (Fig. 2C; Kruskal–Wallis test, χ2 = 7.236, P b 0.01) and 65% (Fig. 2D; Kruskal–Wallis test, χ2 = 7.757, P b 0.01), respectively. Group differences among the sham, GDX and GDX-T rats
Fig. 2. Effects of intranasal administration and subcutaneous injection of T on the amount of walking (A), climbing (B), rearing (C) and sniffing (D). The asterisks indicate significant differences (*P b 0.05, **P b 0.01).
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were found in the amount of walking (Kruskal–Wallis test, χ2 = 16.107, P b 0.01), climbing (one-way ANOVA, F(2,21) = 12.129, P b 0.01), rearing (Kruskal–Wallis test, χ2 = 13.528, P b 0.01) and sniffing (Kruskal–Wallis test, χ2 = 14.495, P b 0.01). The post hoc test indicated that the amount of walking, climbing, rearing and sniffing in GDX rats was significantly lower than in the sham rats by 70% (Fig. 2A; P b 0.01), 57% (Fig. 2B; P b 0.01), 68% (Fig. 2C; P b 0.01) and 62% (Fig. 2D; P b 0.01), respectively, and that the amount of above behaviors in GDX rats was restored to the level of sham rats after intranasal administration of T, except for the rearing. The amount of rearing in GDX-T rats was still lower than in the sham rats by 47% (P b 0.05), even though the amount was increased 68% (Fig. 2C; P N 0.05) compared to GDX rats. Thigmotaxic behavior The effect of intranasal administration of T was further tested in terms of the thigmotaxic behavior of rats. No differences were observed for thigmotaxic behavior (data not shown). Motor behaviors Vertical activity, horizontal activity and total path length were then examined in the motor behavior experiments. The intranasal administration of T significantly increased the amount of vertical activity and horizontal activity as well as the total path length in intact rats by 61% (Fig. 3A; one-way ANOVA, F(1,14) = 13.002, P b 0.01), 69% (Fig. 3B; Kruskal–Wallis test, χ2 = 6.353, P b 0.05) and 75% (Fig. 3C; one-way ANOVA, F(1,14) = 10.884, P b 0.01), respectively. Group differences among the sham, GDX and GDX-T rats were found in the
amount of vertical activity (one-way ANOVA, F(2,21) = 16.485, P b 0.01), horizontal activity (Kruskal–Wallis test, χ2 = 12.214, P b 0.01) and in the total path length (one-way ANOVA, F(2,21)= 14.446, P b 0.01). The post hoc test found that the amount of vertical activity and horizontal activity in GDX rats was significantly lower than in the sham rats by 60% (Fig. 3A; P b 0.01) and 60% (Fig. 3B; P b 0.01), respectively. The total path length in GDX rats was significantly less than in the sham rats by 59% (Fig. 3C; P b 0.01) and the amount of vertical activity and horizontal activity as well as the total path length in GDX rats were restored to the level of sham rats after intranasal administration of T.
Grooming behaviors Finally, grooming behaviors were tested. No differences were observed for the latency of grooming (data not shown). The intranasal administration of T significantly increased the number of grooming events and the duration of grooming in intact rats by 72% (Fig. 4A; one-way ANOVA, F(1,14) = 13.138, P b 0.01) and 61% (Fig. 4B; oneway ANOVA, F(1,14) = 8.755, P b 0.01), respectively. Group differences among the sham, GDX and GDX-T rats were found in the number of grooming events (Kruskal–Wallis test, χ2 = 13.598, P b 0.01) and the duration of grooming (one-way ANOVA, F(2,21) = 8.658, P b 0.01). The post hoc test revealed that the number of grooming events in GDX rats was significantly less than in the sham rats by 54% (Fig. 4A; P b 0.01), the duration of grooming in GDX rats was significantly shorter than in the sham rats by 55% (Fig. 4B; P b 0.01) and the number of grooming events as well as the duration of
Fig. 3. Effects of intranasal administration and subcutaneous injection of T on the amount of vertical activity (A) and horizontal activity (B) and total path length (C). The asterisks mark significant differences (*P b 0.05, **P b 0.01).
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Fig. 4. Effects of intranasal administration and subcutaneous injection of T on the number of grooming events (A) and the duration of grooming (B). The asterisks indicate significant differences (*P b 0.01).
grooming in GDX rats was restored to the level of sham rats after intranasal administration of T. The effect of subcutaneous injection of testosterone on open field behavior Significant decreases in open field activity were observed in GDXsc rats (Figs. 1–4). The subcutaneous injection of T only restored the amount of walking (Fig. 2A), horizontal activity (Fig. 3B) and the total path length (Fig. 3C) to the level of sham-sc rats. There were no significant differences between GDX-sc.T and GDX-sc rats in the number of immobile-sniffing events, the amount of climbing, rearing, vertical activity, the number of grooming events and the duration of grooming, except for the amount of sniffing (Fig. 2D). In total, we observed 12 kinds of behavioral operations in the open field test (Table 1). The open field activity scores for most tests significantly increased with intranasal delivery of T in GDX rats and intact rats. Intranasal administration of T could restore the most of parameters of open field behavior in GDX rats, however, only a few behavioral operations were improved in GDX-sc rats after subcutaneous T administration treatment. Discussion In the present study, we found that the parameters of immobilesniffing, exploratory behavior, motor behavior and grooming behavior in rats were significantly reduced after the rats were gonadectomized.
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Intranasal administration of T to GDX rats and intact rats increased the above behaviors. The most of parameters of open field behavior were improved in GDX-T rats. Our results indicated that chronic nasal administration of T could affect the central nervous system of rats. It was found that the T levels in the blood after nasal and intravenous administration were similar in rats and the bioavailability of the nasally administered T was over 90% (Hussain et al., 1984). In mice, the bioavailability in serum was found to be 75% after intranasal administration (Banks et al., 2009). Testosterone enters the brain through direct and indirect transport routes after intranasal administration. About two thirds of T in the brain after intranasal administration directly entered the brain from the nasal area without first being absorbed into the blood and the remainder indirectly entered the brain by first entering the blood and then crossing the blood–brain barrier (BBB) (Banks et al., 2009). In our study, the intranasal administration of T could improve the most of the reduced parameters of open field behavior in GDX rats, but, only a few behavioral parameters were improved in GDX-sc rats when T was given subcutaneously at same dose. These results in the present experiments suggested that intranasal administration of T is a more efficacious way of targeting the brain, which was inferred from a recent study that intranasal administration of T at the dose of 2.0 mg/ kg could effectively increase dopaminergic activity in the neostriatum and nucleus accumbens, however, subcutaneous administration of T at the same dose did not lead to an increase in dopamine in the neostriatum and nucleus accumbens and the increased dopamine was only seen at higher doses of 8.0 mg/kg in the neostriatum (de Souza Silva et al., 2009). Intranasal administration of T produced a targeted delivery to the brain, especially to the olfactory bulbs, hypothalamus, striatum, and hippocampus (Banks et al., 2009). The difference of regional brain distribution of T after intranasal administration might be related to the regional differences in androgen receptor binding. Androgen receptor mRNA-containing neurons were heavily distributed in the olfactory bulbs, hypothalamus and hippocampus (Simerly et al., 1990). The difference in terms of the effect potency of the intranasal delivery of T on the behaviors of rats between GDX-T vs. GDX and intact-T vs. intact in our study is probably due to the different serum T levels in GDX (the absence of endogeneous gonadal hormones) and intact (the presence of endogeneous gonadal hormones) rats. There are two factors that should be further investigated in the future studies on the effects of the intranasal administration of T on the open field behavior. One is T dose delivered intranasally to GDX rats. The dose in the present study was chosen on the basis of the findings that the intranasal application of 2.0 mg/kg of T resulted in an increase in dopamine levels in both the neostriatum and nucleus accumbens (de Souza Silva et al., 2009). Intranasal delivery of 2.0 mg/ kg/day of T to GDX rats improved the most of parameters of open field behavior but did not fully restore the reduced behaviors in GDX rats. Different doses should be further tested in the following study. Another is sex hormone-binding globulin, which limits T ability to enter the brain from blood stream and is absent in rodent animals (Downer et al., 2001). Studies of the effect of intranasal delivery of T on the open field behavior should be further performed in non-rodent species. Testosterone can easily cross BBB due to its lipophilic properties, but systemic administration of T has been shown to be problematic. The efficacy of oral testosterone undecanoate is limited because of its unreliable oral bioavailability, fluctuating serum levels and short halflife necessitating multiple daily doses. Testosterone taken orally is also rapidly inactivated by first-pass hepatic metabolism, which makes oral therapy an ineffective means of delivering T. Deep intramuscular injections are invasive and can cause patient discomfort (Nieschlag et al., 2004). The insertion of T implants requires minor surgery and can be painful, and extrusion of the pellets is common (Handelsman et al., 1997). Transdermal T non-scrotal patches result
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in skin irritation in patients (Parker and Armitage, 1999; Jordan, 1997). Testosterone gels are not always considered convenient and bear the risk of skin-to-skin transfer (Rolf et al., 2002). The ideal T replacement therapy should offer safety, efficacy, cost-effectiveness, convenience, a good release profile, dosing flexibility, and effective normalization of T levels (Nieschlag et al., 2004). Targeting T to the central nervous system should allow the administration of lower doses of T and fewer peripheral side effects. The intranasal route would seem to be advantageous for T administration. Lipophilic substances, such as T, are transported directly from the nose to the brain through: (1) the olfactory epithelial pathways (across the sustentacular cells, through tight junctions between sustentacular cells, or clefts between sustentacular cells and olfactory neurons), (2) the olfactory nerve pathway, or (3) through the trigeminal nerve pathway (Graff and Pollack, 2005; Illum, 2004, 2007; Thorne et al., 2004). This delivery route would also negate the effects of serum protein binding so that the intranasal route dose of T needed for brain delivery would likely be lower than the systemic dose (Morley et al., 2002). Testosterone was administered intranasally in anesthetized male rats, and its effects on the activity of dopaminergic and serotonergic neurons in the neostriatum and nucleus accumbens were assessed by means of microdialysis and high performance liquid chromatography (HPLC). The intranasal administration of T led to an immediate increase in DA and 5-HT levels in both the neostriatum and nucleus accumbens (de Souza Silva et al., 2009), which indicated that intranasal administration of T rapidly activated central dopaminergic and serotonergic systems. Increased behavioral scores in the open field test after chronic intranasal delivery of T might result from the altered dopaminergic and serotonergic system. Immunocytochemical (DonCarlos et al., 1991; Kritzer, 1997) and in situ hybridization (Simerly et al., 1990) studies identified subpopulations of intracellular gonadal hormone receptor-bearing neurons in the substantia nigra (SN) and the ventral tegmental area (VTA), which suggest that specific subsets of midbrain neurons might be direct targets of gonadal hormones. It was found that nearly every androgen receptor bearing cell in the VTA and SN pars compacta, roughly half in the SN pars lateralis, and about one-third in the retrorubral fields were tyrosine hydroxylase immunopositive and these androgen receptors tend to occupy regions labeled by injections in limbic or cortical targets (Kritzer, 1997; Creutz and Kritzer, 2004). Chronic intranasal administration of T might result in transcriptional changes in protein synthesis via intracellular androgen receptors in the mesolimbic and mesocortical DA neurons and influence behavioral functions. Lower androgenic level or T deficit influenced organism. Testosterone deficiency disorder could refer to premature mortality and to a number of co-morbidities, such as sexual disorders and diabetes as well as metabolic syndrome. Testosterone deficiency occurs mainly in ageing men when prostate disease starts to emerge. The current T supplements by different route of administration developed during the last decade of these patients (Raynaud, 2009). When intranasal administration of T is also expected to be used as adjunctive therapy for some neuropsychiatric disorders, such as PD (Mitchell et al., 2006), the direct nasal route of T delivery to brain would become the dominant factor in determining brain uptake because the ability of T to enter the brain is limited in the presence of sex hormone-binding globulin in serum (Downer et al., 2001; Hobbs et al. 1992; Pardridge et al. 1980). Gonadectomy in male rats decreased immobile-sniffing, exploratory behavior, motor behavior and grooming behavior. There is not a single supplement that can treat it all. The impaired behaviors insulted by gonadectomy could be mostly restored by chronic intranasal administration of T, which is better than subcutaneous injection of T, demonstrating that T can be delivered to the brain to affect the brain functions by intranasal administration. Therefore, the intranasal T delivery is an effective option for targeting the central
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