Neurochem Res (2010) 35:1302–1314 DOI 10.1007/s11064-010-0183-6
ORIGINAL PAPER
Intranasal Administration of Nerve Growth Factor Produces Antidepressant-Like Effects in Animals Cui-ge Shi • Lu-ming Wang • Ying Wu • Peng Wang • Zhu-jun Gan • Kai Lin • Li-xin Jiang • Zhi-qing Xu • Ming Fan
Accepted: 23 April 2010 / Published online: 3 June 2010 Ó Springer Science+Business Media, LLC 2010
Abstract Many works showed that nerve growth factor (NGF) injected into the brain of animal model emerges potential antidepressant effects. However, this route of administration significantly restricts the application of NGF clinically. Here, we reported that intranasal NGF could provide an alternative to intraventricular injection. The behavioral analysis showed that intranasal administration of NGF reduced the immobility time in forced swimming test (FST) and tail suspension test (TST) in mice. Likewise, intranasal NGF increased the sucrose intake and the locomotor activity in rats after unpredictable chronic mild stress (UCMS). Furthermore, intranasal NGF increased the levels of monoamine neurotransmitters (norepinephrine, dopamine) in the frontal cortex and hippocampus and affected the number of 5-bromodeoxyuridine (BrdU), c-fos and caspase-3 positive neurons in dentate gyrus of hippocampus in rats after UCMS. In summary, intranasal NGF had significant antidepressant effects on animal models of depression and this route of administration may provide a promising way to deliver NGF to brain in a therapeutic perspective.
C. Shi P. Wang Z. Gan Z. Xu M. Fan Department of Neurobiology, Capital Medical University, Beijing, China L. Wang Y. Wu M. Fan (&) Department of Brain Protection and Plasticity, Institute of Basic Medical Sciences, 27 Taiping Road, 100850 Beijing, China e-mail:
[email protected] K. Lin Clinic Laboratory Center, Air Force Genneral Hospital, Beijing, China L. Jiang Staison Bio-Pharmaceutical Company, Beijing, China
123
Keywords NGF Intranasal delivery Depression Animal models Abbreviations NGF Nerve growth factor NTFs Neurotrophic factors 5-HT Serotonin NE Norepinephrine DA Dopamine AMI Amitriptyline UCMS Unpredictable chronic mild stress CNS Central nervous system BrdU 5-Bromodeoxyuridine DG Dentate gyrus GCL Granule cell layer SGZ Subgranular zone
Introduction Depression causes neurological impairment, including cognitive and motor dysfunction. Neurotrophins are a class of proteins that serve as survival factors for selected populations of central nervous system (CNS) neurons [1]. Recently, a neurotrophin hypothesis of depression has been raised because of following reasons: I. neurodegenerative symptoms occur in depression, which is associated with a decrease in blood or brain concentrations of neurotrophic factors (NTFs); II. antidepressant treatments increase the expression of NTFs; III. NTFs can protect noradrenergic and serotonergic neurons; IV. glucocorticoids may suppress NTF synthesis [2]. Nerve growth factor (NGF) is a neurotrophin which plays an important role in the survival and function of
Neurochem Res (2010) 35:1302–1314
neurons. Decreased NGF has been reported in several animal models of depression [3]. These studies have identified adaptations of intracellular signaling proteins and target genes that could contribute to the action of antidepressant treatment. One target gene of antidepressant treatment is NGF. Antidepressant treatment increases the expression of NGF in limbic structures, most notably the hippocampus [4]. Upregulation of NGF occurs in response to chronic but not acute antidepressant treatment, consistent with the time course for the therapeutic action of antidepressants. The possibility that NGF is also involved in the pathophysiology of stress related mood disorders is supported by reports that NGF expression is decreased by exposure to stress [5]. Clinical brain-imaging studies demonstrate that the volume of the hippocampus is decreased in depressed patients, consistent with the possibility of reduced neurotrophic factor support or synaptic remodeling in depression [6, 7]. Currently, NGF therapy is administrated by intravenous injection and intraventricular infusion. However, these routes of administration significantly restrict the application of NGF clinically [8, 9]. Recently, reviews have shown that many peptides and regulatory proteins can be delivered to the brain by intranasal administration. Additionally, insulin, melanocytestimulating hormone and arginine vasopressin could be delivered to brain. These substances occurred in the cerebrospinal fluid within minutes [10]. This reinforces the idea that even if a substance can cross the blood–brain barrier after intravenous administration, intranasal administration results in much higher brain/serum ratios. This, in turn, would favor effects in the CNS rather than effects at peripheral tissues. So, intranasal administration was a simple, inexpensive, and noninvasive method. The forced swimming test (FST) and tail suspension test (TST) are two widely accepted behavioral models for assessing pharmacological anti-depressant activity. Animal immobility is used as the characteristic behavior in these tests, reflecting the despair as seen in human depression [11]. Likewise, unpredictable chronic mild stress (UCMS) is thought to precipitate or exacerbate several neuropsychiatric disorders including depression and widely used for the simulation of depressive state to estimate the effectiveness of antidepressants in preclinical studies. In addition, the monoamine theory suggests that depression is due to an imbalance of the levels of the monoamine neurotransmitters in the central nervous system and NGF could influence the levels of the monoamine neurotransmitters [12]. In the current study, we assessed the possible antidepressant-like effects of intranasal NGF in three depression models: FST, TST in mice and UCMS in rats. Furthermore, the regulation of monoamine neurotransmitters and neuroprotection induced by intranasal NGF were
1303
examined with the goal of identifying therapeutic strategies against depression in the future.
Experimental Procedure Animals Adult male Kun-Ming (KM) mice weighting 18–22 g and Adult male Sprague–Dawley (SD) rats with weights of 180–220 g were used. All animals were housed 5 per cage under standardized light/dark cycle condition (lights on at 7:00 AM, lights off at 7:00 PM) at a room temperature of 24 ± 1°C and humidity of 60 ± 10% with food and water ad libitum. All experiments were performed in compliance with the ‘Guide for the Care and Use of Laboratory Animals’ of the Institute of Laboratory Animal Resources, National Research Council, and with approval of Beijing Institute of Basic Medical Sciences on Animal Welfare. Materials Amitriptyline (AMI), Serotonin (5-HT), Norepinephrine (NE), and Dopamine (DA) were all purchased from Sigma–Aldrich. NGF was devoted by Staison Bio-Pharmaceutical Company. Following primary antibodies were used in immunohistochemistry: anti-BrdU (ZM-0013; Zhongshan Golden Bridge Biotechnology Company, CHN), anti-Caspase-3 (PR-0284; Zhongshan Golden Bridge Biotechnology Company, CHN) and anti-C-fos (sc-52; Santa Cruz Biotechnology, Santa Cruz, CA).
NGF Nasal Delivery The protocol was modified by De Rosa [13]. For the mice, NGF solution in PBS (0.1 mg/ml) was administered intranasally at the dose of 75 lg/kg, 2.5 ll at a time, alternating the nostrils, with a lapse of 2 min between each administration, for a total of 10 times. The dose of NGF used in rats was used according to the transfer coefficient of the dose from mouse to rat (0.67) multiplied the dose of NGF in mice. For the rats, 1% mebumal sodium was dissolved in 0.9% NaCl was injected i.p. at the dosage of 40 mg/kg of body weight to induce anesthesia. Then, rats were laid on their back, with the head in upright position. NGF solution in PBS (0.1 mg/ml) were administered intranasally at the dose of 50 lg/kg, 10 ll at a time, alternating the nostrils, with a lapse of 2 min between each administration, for a total of 10 times. During these procedures, the nostrils were always kept open.
123
1304
Neurochem Res (2010) 35:1302–1314
Forced Swimming Test in Mice Mice were placed on an individual basis into a Plexiglas cylinder (20 cm height 9 10 cm diameter) which was filled with water (23–25°C) to a depth of 10 cm. This depth was sufficient to keep the mice from supporting themselves by placing their paws or tails on the base of the cylinder. All drugs were administered 60 min prior to testing. A mouse was judged to be immobile when floated in the water in an upright position and made only small movements to keep its head above water in 6 min. During 6-min test session, the duration of immobility was recorded during the last 4 min [14]. The experiment was performed by double-blinded method. Tail Suspension Test in Mice The tail suspension test was carried out as described previously [15]. One hour after drugs administration, mice were suspended by the tail to a horizontal ring stand bar (distance from floor 25 cm) using adhesive tape (distance from tip of tail 2 cm). Then the immobility time was measured. During 6-min test session, the duration of immobility was recorded during the last 4 min. The experiment was performed by double-blinded method. Unpredictable Chronic Mild Stress in Rats The stress protocol was modified by Lu [11]. This paradigm was designed to maximize unpredictability. It consisted of a variety of stressors applied randomly and at varying times of day for 14 days (Table 1). The following stressors were used: (1) 24 h of food deprivation; (2) 24 h of water deprivation; (3) 1–1.5 h of restraint; (4) 10–20 min inescapable foot shock; (5) 5 min warm swim Table 1 The procedure of UCMS
123
(42°C); (6) 5 min cold swim (16°C); (7) 1 min tail clipping. Restraint was performed by placing the animal in a 21 9 6 cm plastic tube and adjusting it with plaster tape on the outside so that the animal was unable to move. There was a 6 cm hole at the far end for breathing. Forced swimming was performed by placing each animal in a glass tank measuring 44 9 33 9 30 cm with 22 cm of water depth. All stressors were applied to animals in a separate procedure room. Control rats were handled daily in the housing room for 14 days.
Sucrose Intake The sucrose preference test consisted of a two-bottle choice paradigm, performed under red light at the beginning of the dark phase. At the start of the experiment, rats were habituated to drink a 1% sucrose solution for 3 days. On day 4, the sucrose solution was replaced with tap water for an additional 2 days Two hours (5:00 P.M.) before the test, rats were singly housed with access to food. At the start of the dark phase (7:00 P.M.), rats were given access to the two bottles (containing water or 1% sucrose). The position of the sucrose bottle (left or right) was balanced between the experimental groups. Fluid intake was then measured for 1 h and the total amount of fluid (water or sucrose) intake was calculated. The preference for sucrose over water was used as a hedonic measure.
Locomotor Activity Locomotor activity was measured by the open field test in a square arena (100 9 100 9 45 cm). The open-field behavior box was divided visually into 25 zones of 20 9 20 cm.
Day
Type of stressor
Time
1
60 min restraint
am
2
20 min inescapable foot shock (1.0 mA, 15, 60 s off)
pm
3
24-h food deprivation
4
5 min warm swim (42°C)
am
5 6
24-h food deprivation 5 min cold swim (16°C)
pm
7
1 min tail clipping (a clothespin placed at 1 cm from the base of tail)
am
8
24-h food deprivation
9
10 min inescapable foot shock (1.0 mA, 15, 60 s off)
pm
10
5 min warm swim (42°C)
am
11
90 min restraint
pm
12
1 min tail clipping (a clothespin placed at 1 cm from the base of tail)
am
13
5 min cold swim (16°C)
pm
14
24-h food deprivation
Neurochem Res (2010) 35:1302–1314
1305
All testing was done in an isolated room between 8:00 A.M. and 12:00 A.M.The protocol was modified by Lu [11]. Spontaneous horizontal activity and vertical activity (or rearing) were measured after UCMS. Briefly, after each rat acclimatize chamber for a period of 2 min, locomotion activity was recorded in terms of total zone numbers of ambulation and rearing (total counts) during a 3-min test.
the concentration of the mix samples was determined, and the known concentration of samples was subtracted from which. The recovery rate is a ratio of the D-value to the concentration of standard solution.
Monoamine Analysis
BrdU is a thymidine analog that is taken up by proliferating cells during the S phase of the cell cycle. Rats were administered BrdU (50 mg/kg, i.p.) twice 1 day for 3 consecutive days and perfused after the last injection.
Rats subjected to UCMS were euthanized by decapitation at the end of the behavioral tests. Brains were quickly removed, and the frontal cortex and hippocampus were dissected out on ice and weighed. Each cerebral region was homogenized and centrifuged at 3,000 rpm for 10 min with 30 times (w/v) n-butanol, the supernatant was stored at -20°C until use. The contents of Norepinephrine (NE), Dopamine (DA) and Serotonin (5-HT) were measured by chemiluminescent assay (Fluorospectrophotometer RF-5301PC, Shimadzu, JPN). For assay of NE and DA [16], 1.5 ml n-butanol supernatant was homogenized in 2.1 ml PBS and 0.2 ml 1 mM HCl, with 5-min oscillations, then centrifuged at 1,500 rpm for 5 min. 2.0 ml Water phase was detected and homogenized in 0.25 ml 4%EDTA, 0.2 ml Iodine reagent, 0.25 ml 2.5% Na2SO3 and 0.3 ml 5 M ethanoic acid, then boiled 10 min. After cooling, the fluorescence NE intensity was measured using a spectrofluorometer with 385 nm excitation and 480 nm emission wavelength (2 nm band width). Then, all tubes added 0.4 ml phosphoric acid, boiled 5 min. After cooling, the fluorescence DA intensity was measured by a spectrofluorometer with 325 nm excitation and 375 nm emission wavelength. For assay of 5-HT [17], 1 ml n-butanol supernatant was homogenized in 2.5 ml n-heptane, 1 ml 0.1 M HCl and 0.2 ml 0.01 M HCl, with 10-min oscillations, then centrifuged at 1,500 rpm for 5 min. 1 ml Water phase was detected and homogenized in 0.1 ml 1% cysteine, 1.5 ml OPT (80 mg/L HCl) and 0.1 ml 0.02% NaIO4, then boiled 10 min. After cooling, the fluorescence 5-HT intensity was measured by a spectrofluorometer with 365 nm excitation and 480 nm emission wavelength. Fluorescence was expressed as fluorescence units. The actual concentration of the NE, DA and 5-HT was calculated by comparison with the standard curves. Results are expressed as lg/g tissue.
BrdU Administration
Tissue Preparation Rats were deeply anesthetized with mebumal sodium and perfused transcardially with 0.9% NaCl for 5 min, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) for 20 min. Then, the brains were removed, postfixed in 4% PFA, and embedded in paraffin. Threemicrometre-thick paraffin sections were cut from the hippocampus. Immunohistochemistry The immunohistochemical appearance of the BrdU, C-fos and Caspase-3 was successfully optimized on paraffin section. Briefly, paraffin sections were deparaffinised and hydrated through xylene and graded alcohol series, respectively. After rinsing with water, sections were boiled in 0.1 M citric acid (pH 6.1) for 10 min and allowed to cool down to room temperature. Sections were washed with PBS and placed in 0.3% H2O2 to quench endogenous peroxidase activity, and washed again. Sections were incubated with normal blocking serum for 1 h and then with anti-BrdU (1:100), anti-c-fos (1:300) and anti-Caspase-3 (1:300) antibody overnight respectively. After washing, sections were incubated for 1 h with biotinylated secondary antibody followed by incubation with a preformed complex of avidin and biotinylated peroxidase. Sections were incubated in peroxidase substrate solution (Diaminobenzidine Tetrahydrochloride, DAB) until desired stain intensity developed, rinsed with water, cleared and mounted. Statistical Analyses
Recovery rate of Monoamine Analysis The recovery rate is a standard parameter in terms of biochemical analysis. Different samples with a known amount of NE, DA and 5-HT were added into NE, DA and 5-HT standard solution with a known amount, respectively,
For statistical analysis, a standard software package (SAS 10.0) was used. All data are presented as means ± SEM. Differences between groups were compared by Dunnett test. Statistical significance was set at P \ 0.05. Quantitative assessment of BrdU, c-fos and caspase-3 positive
123
1306
Neurochem Res (2010) 35:1302–1314
Results Immobility time in FST and TST Intranasal NGF at the doses of 75 lg/kg produced a reduction in the immobility time in FST and TST in mice (Fig. 1). In FST (Fig. 1a), behavioral analysis revealed that the immobility time in the control group was 98.1 ± 8.0 s, NGF pretreatment (60 min before test) significantly reduced the immobility time to 51.3 ± 5.1 s (P = 0.002 vs. control, n = 8). As a positive control, oral administration of antidepressant drug Amitriptyline (AMI, 15 mg/kg) also decreased the immobility time to 33.5 ± 6.0 s (P = 0.005 vs. control, n = 8). In TST (Figs. 1b, 2), the immobility time in the control group was 77.5 ± 10.1 s, NGF pretreatment (60 min before test) significantly reduced the immobility time to 28.1 ± 6.2 s (P = 0.005 vs. control, n = 8), oral administration of antidepressant
A
Immobility(sec)
#
#
25
*
20 15 10 5 0
**
Con
UCMS
NGF AMI +UCMS
30 40
Con
NGF
AMI
90
TST 60
**
Con
NGF
30 #
AMI
Fig. 1 Effects of intranasal NGF (75 lg/kg) on the immobility time in FST (a) and TST (b) in mice. As a positive control, Amitriptyline (AMI, 15 mg/kg, po.) was administered once. The test was performed 1 h after the last administration of NGF or AMI. Vertical bars represent means ± SEM. * P \ 0.05, ** P \ 0.01 vs. control. n = 8
#
20
**
10
0
**
30
0
123
One of the depression-like phenotypes induced by UCMS is decreased sucrose preference, considered a sign of the hedonic deficit seen in depressed patients. Intranasal NGF remarkably increased sucrose preference in rats subjected to UCMS (Fig. 3a–b). The total mounts of sucrose intake and sucrose versus water ratio of the rats were 21.6 ± 2 ml and 33 ± 2.4 in control group, which were much higher than those subjected to UCMS (18.2 ± 2.8 ml and 9.8 ± 0.9, P = 0.022 and 0.008 vs. control, respectively, n = 8). During the 2-week treatment period of NGF, the total mounts of sucrose intake and sucrose versus water ratio increased to 23.5 ± 3.3 ml and 17.6 ± 1.6 (P = 0.013 and 0.033 vs. UCMS, respectively, n = 8). As a positive control, AMI (10 mg/kg) increased the total mounts of sucrose intake and sucrose versus water ratio to 24 ± 2.3 and 23.1 ± 2.1, respectively (P = 0.017 and 0.027 vs. UCMS, n = 8).
**
60
0
B
FST
Sucrose Preference
Sucrose preference (Sucrose : Water ratio)
Immobility(sec)
90
drug AMI (15 mg/kg) also decreased the immobility time to 40.2 ± 8.1 s (P = 0.02 vs. control, n = 8).
Sucrose intake(ml)
neuron was achieved by counting the number of positive cells per square millimeter in selected brain areas. All slides were numbered without the treatment information to avoid any bias in counting. Cells with distinct brown nuclear immunoreactivity staining in various brain regions were manually counted under light microscopy with low magnification (9100) with the aid of a 1 mm2 ruler.
Con
UCMS
NGF AMI +UCMS
Fig. 2 Effect of intranasal NGF on UCMS induced reduction in sucrose preference. a Total amount of sucrose intake. b Sucrose preference expressed as a ratio of the volume of sucrose intake to the volume of water intake. UCMS rats display a substantially decreased sucrose preference. This effect of UCMS was reversed by intranasal NGF (50 lg/kg). As a positive control, AMI (10 mg/kg, po.) was given for 14 days. Vertical bars represent means ± SEM. * P \ 0.05, ** P \ 0.01 vs. control. # P \ 0.05, ## P \ 0.01 vs. UCMS. n = 8
1307
60
#
*
60
Y= -1.71143+0.16564X R=0.998
40
20
0 0
0
Con
UCMS
NGF AMI +UCMS
#
#
15
*
10
50
100
150
200
250
300
350
400
Concentration of NE(ng/ml)
B
80
Light intensity
activity(counts/3 min)
80
20
B
Vertical
#
40
A
60
Y= 1.03429+ 0.1609X R=0.996
40
20
0
5
0
50
100 150 200 250 300 350 400
Concentration of DA(ng/ml) 0
C 120 Con
UCMS
AMI NGF +UCMS
Fig. 3 Effects of intranasal NGF (50 lg/kg) on the horizontal activity (a) and vertical activity (b) in rats after UCMS. As a positive control, AMI (10 mg/kg, po.) was given for 14 days. Vertical bars represent means ± SEM. * P \ 0.05, ** P \ 0.01 vs. control. # P \ 0.05, ## P \ 0.01 vs. UCMS. n = 8
Locomotor Activity
Light intensity
Horizontal activity(counts/3 min)
A
Light intensity
Neurochem Res (2010) 35:1302–1314
Y= -0.26286+0.24745X R=0.998
90
60
30
0 0
50
100 150 200 250 300 350 400
Concentration of 5-HT (ng/ml)
Intranasal NGF remarkably increased the locomotor activity in rats subjected to UCMS (Fig. 3a–b). The horizontal activity and vertical activity of the rats were 58.8 ± 6.2 and 14.8 ± 2.5 counts/3 min in control group, which were much higher than those subjected to UCMS (23.7 ± 4.8 and 6.8 ± 2.4 counts/3 min, P = 0.001 and 0.034 vs. control, respectively, n = 8). During the 2-week treatment period of NGF, the horizontal activity and vertical activity increased to 38.7 ± 3.9 and 14.2 ± 2.1 counts/3 min (P = 0.032 and 0.043 vs. UCMS, respectively, n = 8). As a positive control, AMI (10 mg/kg) increased horizontal activity and vertical activity significantly to 39.2 ± 4.1 and 15.5 ± 1.7 counts/3 min, respectively (P = 0.036 and 0.011 vs. UCMS, n = 8). Monoamine Levels in Brain Tissues The standard curves of NE, DA and 5-HT were showed in Fig. 4a–c. The assay was linear in the range 0 to 400 ng/ml of NE, DA and 5-HT in cuvette, respectively. The levels of NE, DA and 5-HT in the frontal cortex and hippocampus
Fig. 4 Relation between increase of fluorescence and concentration of NE (a), DA (b) and 5-HT (c) in 3 ml samples. Results are based on duplicate assays and corrected for reagent blank
were observed and calculated by standard curve equation (Fig. 5). The mean NE levels in the frontal cortex and hippocampus in control group were 3.16 ± 0.49 and 4.15 ± 0.22 lg/g tissue, respectively, which were much higher than those subjected to UCMS (1.81 ± 0.42 and 2.43 ± 0.60 lg/g tissue, P = 0.026 and 0.038 vs. control, respectively, n = 8). As expected, NE levels in the frontal cortex and hippocampus after intranasal NGF were increased to 2.94 ± 0.76 and 4.05 ± 0.70 lg/g tissue (P = 0.032 and 0.034 vs. UCMS, respectively, n = 8). Under the same experimental conditions, NE levels in the frontal cortex and hippocampus after AMI treatment were increased to 3.28 ± 0.43 and 4.16 ± 0.16 lg/g tissue (P = 0.002 and 0.018 vs. UCMS,respectively, n = 8, Fig. 5a).
123
1308
Neurochem Res (2010) 35:1302–1314
A
cortex hippocampus
6.0
NE ug/g tissue)
# #
4.5
*
#
##
3.0
* 1.5
0.0
Con
UCMS
AMI
NGF +UCMS
B
cortex hippocampus
4
DA(ug/g tissue)
#
#
3 #
* 1
0
Con
UCMS
AMI
NGF +UCMS
cortex hippocampus
C
#
5-HT ug/g tissue
2.5
#
2.0
* *
1.5 1.0 0.5 0.0
Con
UCMS
NGF
AMI
+UCMS
Fig. 5 Effects of intranasal NGF (50 lg/kg) on the levels of NE (a), DA (b) and 5-HT (c) in cortex and hippocampus in rats subjected to UCMS. As a positive control, AMI (10 mg/kg, po.) was given for 14 days. Vertical bars represent means ± SEM. * P \ 0.05. ** P \ 0.01 vs. control. # P \ 0.05, ## P \ 0.01 vs. UCMS. n = 8
The mean DA levels in the frontal cortex and hippocampus in control group were 1.67 ± 0.42 and 2.90 ± 0.59 lg/g tissue, respectively, which were much higher than those subjected to UCMS (0.98 ± 0.39 and 1.60 ± 0.35 lg/g tissue, P = 0.028 and 0.031 vs. control, respectively, n = 8). As expected, DA levels in the frontal cortex and hippocampus after intranasal NGF were increased to 2.11 ± 0.49 and 2.72 ± 0.71 lg/g tissue (P = 0.027 and 0.010 vs. UCMS, n = 8). Under the same experimental conditions, DA levels in the frontal cortex and hippocampus after AMI treatment were increased to 2.08 ± 0.43 and 2.80 ± 0.61 lg/g tissue (P = 0.024 and 0.040 vs. UCMS, n = 8, Fig. 5b).
123
Recovery Rate of Monoamine Analysis
#
*
2
The mean 5-HT levels in the frontal cortex and hippocampus in control group were 2.20 ± 0.23 and 1.98 ± 0.30 lg/g tissue, respectively, which were much higher than those subjected to UCMS (1.21 ± 0.30 and 1.37 ± 0.23 lg/g tissue, P = 0.021 and P 0.026 vs. control, n = 8). As expected, 5-HT levels in the frontal cortex and hippocampus after intranasal NGF were increased to 1.80 ± 0.33 and 1.39 ± 0.29 lg/g tissue (P = 0.071 and 0.083 vs. UCMS, n = 8). Under the same experimental conditions, 5-HT levels in the frontal cortex and hippocampus after AMI treatment were increased to 2.01 ± 0.39 and 1.97 ± 0.47 lg/g tissue (P = 0.042 and 0.032 vs. UCMS, n = 8, Fig. 5c).
NE, DA and 5-HT samples with 5, 10, 20 and 30 ng/ml were added into 50 ng/ml standard solution of NE, DA and 5-HT, respectively, the concentration of the mix samples was determined and the recovery rate were calculated. The results showed that the recovery rate of NE, DA and 5-HT were 100.7 ± 6.4%, 101.3 ± 4.9% and 99.5 ± 5.0%, respectively. Effect of NGF on BrdU-Positive Neurons There were few BrdU? cells in control group (13.6 ± 3.31 cells/mm2). The BrdU? cells increased to 19.2 ± 6.83 cells/mm2 in those subjected to UCMS (P = 0.085 vs. control, n = 6). Intranasal NGF could increase the number of BrdU? cells to 38.3 ± 9.57 cells/mm2 (P = 0.010 vs. UCMS, n = 6). As a positive control, AMI treatment increased the number of BrdU? cells to 37.7 ± 8.94 cells/mm2 (P = 0.004 vs. UCMS, n = 6, Fig. 6). Effect of NGF on c-fos-Positive Neurons There were significant difference between control group and those subjected to UCMS in the number of c-fos ? cells located in the SGZ (42.6 ± 7.31 and 16.3 ± 6.56 cells/mm2, respectively, P = 0.001, n = 6). Intranasal NGF could increase the number of c-fos? cells to 39.5 ± 5.89 cells/mm2 (P = 0.001 vs. UCMS, n = 6). As a positive control, AMI treatment could increase the number of c-fos? cells to 46.1 ± 9.57 cells/mm2 (P = 0.001 vs. UCMS, n = 6, Fig. 7). Effect of NGF on Caspase-3-Positive Neurons The results showed that there were significant difference between control group and those subjected to UCMS in the number of caspase-3? cells located in the SGZ (56.6 ± 14.2 and 76.7 ± 17.5 cells/mm2, respectively,
Neurochem Res (2010) 35:1302–1314
Number of BrdU positive 2 cells cells/mm
E
1309
#
50
##
40
30
20
10
0
Con
UCMS
AMI
NGF
+UCMS
Fig. 6 Newly generated cells in the dentate subgranular zone of a control rat (A1), a rat receiving UCMS (B1), and a rat receiving UCMS and intranasal NGF (C1), a rat receiving UCMS and positive control AMI (D1) visualized through BrdU immunostaining. A2–D2 are magnified views of regions from A1 to D1. A3–D3 are magnified
views of regions from A2 to D2. Scale bars: 100 lm. Bar chart (E) illustrates quantification of BrdU positive cells in the SGZ of the hippocampus. Data are means ± SEM of average no. of cells/mm2 in dentate gyrus. * P \ 0.05. ** P \ 0.01 vs. control. # P \ 0.05, ## P \ 0.01 vs. UCMS. n = 6
P = 0.034, n = 6). Intranasal NGF could decrease the number of caspase-3 ? cells to 57.3 ± 11.1 cells/mm2 (P = 0.038 vs. UCMS, n = 6). Under the same experi-
mental conditions, AMI treatment could decrease the number of caspase-3 ? cells to 71.5 ± 8.02 cells/mm2 (P = 0.52 vs. UCMS, n = 6, Fig. 8).
123
Neurochem Res (2010) 35:1302–1314
E
60
Number of c-fos positive
1310
45
##
cells cells/mm
2
##
30
** 15
0
Con
UCMS
NGF
AMI
+UCMS
Fig. 7 C-fos positive cells in the dentate gyrus of a control rat (A1), a rat receiving UCMS (B1), and a rat receiving UCMS and intranasal NGF (C1), a rat receiving UCMS and positive control AMI (D1). A2-D2 are magnified views of regions from A1 to D1. A3–D3 are magnified views of regions from A2 to D2. Scale bars: 100 lm. Bar
123
chart (E) illustrates quantification of c-fos positive cells in the SGZ of the hippocampus. Data are means ± SD of average no. of cells/mm2 in dentate gyrus. *P \ 0.05. **P \ 0.01 vs. control. # P \ 0.05, ## P \ 0.01 vs. UCMS. n = 6
Neurochem Res (2010) 35:1302–1314
*
100
#
75 2
cells ( cells/mm )
Number of caspase-3 positive
E
1311
50
25
0
Con
UCMS
NGF
AMI
+UCMS Fig. 8 Caspase-3 positive cells in the dentate gyrus of a control rat (A1), a rat receiving UCMS (B1), and a rat receiving UCMS and intranasal NGF (C1), a rat receiving UCMS and positive control AMI (D1). A2–D2 are magnified views of regions from A1 to D1. A3–D3 are magnified views of regions from A2 to D2. Scale bars: 100 lm.
Bar chart (E) illustrates quantification of caspase-3 positive cells in the SGZ of the hippocampus. Data are means ± SEM of average no. of cells/mm2 in dentate gyrus. *P \ 0.05. **P \ 0.01 vs. control. # P \ 0.05, ## P \ 0.01 vs. UCMS. n = 6
123
1312
Discussion These studies evaluated the antidepressant effects of NGF intranasal delivery to the central nervous system (CNS). NGF induces multiple changes in depression models: (1) a reduction in immobility time and an enhancement in motor behavior; (2) an increase in the levels of NE and DA; (3) an increment in cell proliferation; and (4) regulation of the expression of c-fos and caspase-3. Although it is difficult to model complex human behaviors in rodents, there are a number of behavioral paradigms that measure antidepressant activity. Rodents forced to swim in a narrow space from which there is no escape will, after an initial period of vigorous activity, adopt a characteristic immobile posture, making only those movements necessary to keep their heads above the water. It was hypothesized that immobility reflected the animals’ having learned that escape was impossible and their having given up hope. Immobility was therefore given the name ‘‘behavioral despair’’. Immobility was subsequently found to be reduced by a wide range of clinically active antidepressant drugs. This simple behavioral procedure has since become a useful test for screening novel antidepressants. An equivalent procedure in mice is also described along with a ‘‘dry’’ version of the test where immobility is induced simply by suspending the mouse by the tail [18]. The present study provides behavioral evidence for the antidepressant-like activities of NGF. In our studies, acute NGF administration showed a tendency to reduce the immobility time in tail suspension and forced swimming test in mice. The differences were statistically significant compared with the control group. In these models, NGF produced a dramatic reduction of the duration of immobility, with a profile comparable to that observed for the classical antidepressant drug AMI. In the current study one well established model, UCMS, is responsive to chronic antidepressant treatment and has been linked to alterations in hippocampal cytogenesis [19]. The antidepressant-like properties of intranasal NGF were evaluated by measuring the effects of NGF on sucrose preference and behaviors in the UCMS model. Systemic administration of NGF reversed the reduced sucrose preference in chronically stressed rats. This effect is comparable to clinically active antidepressants tested in similar animal models. Further, NGF produced a behavioral effect in locomotor activity. It has been shown that NGF intranasal delivery plays a critical role in mediating responsiveness to UCMS. The results of the current study provide correlative data that intranasal NGF underlies behavioral actions of antidepressants. Monoamine hypothesis proposes that depression results from a CNS deficiency of monoamine (NE, DA and 5-HT) functions: decreasing monoamine synthesis, promoting
123
Neurochem Res (2010) 35:1302–1314
reuptake of monoamines and accelerating breakdown of monoamines. In addition to neurotrophic activity, increasing evidences give rise to possible multifunctional properties of NGF on monoamine system including NE, DA and 5-HT [20, 21]. In this study, we clearly demonstrated that NGF led to mediator release of NE and DA. The possible mechanism might be that NGF could counter the depletion of NE store and induce DA release [22]. Likewise, binding of NGF to TrkA rapidly induces autophosphorylation of tyrosine residue of the receptor, which leads to activation of downstream signal cascades including MAPK and PI3 K, thereby results in chemotactic movement. Simultaneous addition of MAPK kinase and PI3 K inhibitors completely blocked NGF-induced 5-HT release, suggesting that 5-HT release may be mediated through the signal transduction cascades [20]. In our study, intranasal NGF was not capable of releasing 5-HT. In adults, cells grafted to the CNS can survive and integrate but with low efficiency. Recently, it is now well established that antidepressants enhance neurogenesis in the dentate gyrus, perhaps to replace those cells damaged by stress [23]. Indeed, antidepressant treatments can rescue the decrease in proliferation of progenitor cells within the SGZ after depression paradigms. It has been demonstrated that neurogenesis is actually necessary for the behavioral effects of antidepressants in a rodent model of depression using x-irradiation of the hippocampus to block de novo proliferation [24, 25]. BrdU-immunoreactive (BrdU?) cells were distributed throughout the subgranular zone (SGZ) and the dentate gyrus (DG) of both genotypes. As expected, the majority of BrdU? cells were located within the SGZ and the inner half of the granule cell layer (GCL) with fewer cells present in the outer half of GCL or in the hilus. In this study, NGF enhanced neurogenesis in the dentate gyrus of adult male rats. These findings suggest that the survival and differentiation of neural progenitor cells could be improved by intranasal NGF and its effects were similar to those of the classical antidepressant drug AMI. C-fos, a well-established oncogene, is considered to play a critical role in tumorigenesis, proliferation and transformation, angiogenesis, tumor invasion, and metastasis, and its expression is associated with poor clinical outcomes. C-fos protein is a product of the c-fos immediate-early gene and is an intranuclear phosphoprotein, being rapidly transported in the nucleus of the cell after translation. C-fos protein has been used as a marker for the activation of neurons in the brain since electrical or chemical stimulation results in markedly enhanced expression of c-fos mRNA as well as protein expression [26–29]. In this study, NGF enhanced c-fos expression in the dentate gyrus in depression. These findings suggest that both intranasal NGF and AMI could activate neurons after stress.
Neurochem Res (2010) 35:1302–1314
Apoptosis can act as an innate defense that restricts injury spread by eliminating cells and triggering pathology pathways. Caspase-3 is a principal effector caspase in apoptotic cascades leading to neuronal apoptosis, as caspase-3-null mice lose developmental neuronal apoptosis [30]. Because the hippocampus has been shown to be the most vulnerable to depression, we focused on the damage in SGZ of the hippocampus in adult rat brains after UCMS. In control rats, hippocampal pyramidal neurons with pyknotic nuclei were hardly detected, although positive staining for caspase-3 was demonstrated in a small number of pyramidal neurons. Such neuron death was naturally occurring cell death (programmed cell death) that has been shown in the hippocampus of rat brains. In the UCMS model, we confirmed that the death mode of pyramidal neurons in the hippocampus was distinct from necrosis and more pyramidal neurons in SGZ of hippocampus were immunostained for activated caspase-3 after UCMS. In our experiment, intranasal delivery of rats with NGF significantly inhibited caspase-3 expression. AMI did not affect caspase-3 expression,that may be related to its neurotoxicity [31]. These findings suggest that the apoptosis of neural progenitor cells in depression can be interrupted by intranasal NGF. In conclusion, NGF intranasal delivery not only rescues the main behavior hallmarks of depression but also regulates monoamine neurotransmitters and hippocampal cytogenesis. Intranasal NGF could play in the onset and therapy of depression and these results highlight the possibility that the olfactory pathway can be a promising, noninvasive route of administration for the delivery of NGF, allowing a long-term treatment of depression. Acknowledgments The work was supported by the National Key Grant of Basic Research Project (2006CB504100) and the Key Grant of Natural Science Foundation of China (30393130).
References 1. Franke B, Bayatti N, Engele J (2000) Neurotrophins require distinct extracellular signals to promote the survival of CNS neurons in vitro. Exp Neurol 165(1):125–135 2. Vaidya VA, Duman RS (2001) Depression—emerging insights from neurobiology. Br Med Bull 57:61–79 3. Song C, Zhang XY, Manku M (2009) Increased phospholipase A2 activity and inflammatory response but decreased nerve growth factor expression in the olfactory bulbectomized rat model of depression: effects of chronic ethyl-eicosapentaenoate treatment. J Neurosci 29(1):14–22 4. Malberg JE, Eisch AJ, Nestler EJ et al (2000) Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. J Neurosci 20(24):9104–9110 5. Purves D, Nja˚ A (1976) Effect of nerve growth factor on synaptic depression after axotomy. Nature 8 260(5551):535–536 6. Vakili K, Pillay SS, Lafer B et al (2000) Hippocampal volume in primary unipolar major depression: a magnetic resonance imaging study. Biol Psychiatry 47(12):1087–1090
1313 7. Videbech P, Ravnkilde B (2004) Hippocampal volume and depression: a meta-analysis of MRI studies. Am J Psychiatry 161:1957–1966 8. Shoemaker SE, Kudwa AE, Isaacson LG (2002) Sympathetic ingrowth to the trigeminal ganglion following intracerebroventricular infusion of nerve growth factor. Brain Res 956(1):136– 148 9. Silver MA, Fagiolini M, Gillespie DC et al (2001) Infusion of nerve growth factor (NGF) into kitten visual cortex increases immunoreactivity for NGF, NGF receptors, and choline acetyltransferase in basal forebrain without affecting ocular dominance plasticity or column development. Neuroscience 108(4):569–585 10. Benedict C, Hallschmid M, Hatke A et al (2004) Intranasal insulin improves memory in humans. Psychoneuroendocrinology 29(10):1326–1334 11. Lu XY, Kim CS, Frazer A, Zhang W (2006) Leptin: a potential novel antidepressant. Proc Natl Acad Sci U S A 103(5):1593– 1598 12. Amino S, Itakura M, Ohnishi H et al (2002) Nerve growth factor enhances neurotransmitter release from PC 12 cells by increasing Ca2?-responsible secretory vesicles through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase. J Biochem 131:887–894 13. De Rosa R, Garcia AA, Braschi C et al (2005) Intranasal administration of nerve growth factor (NGF) rescues recognition memory deficits in AD11 anti-NGF transgenic mice. Proc Natl Acad Sci U S A 102(10):3811–3816 14. Ushijima K, Sakaguchi H, Sato Y et al (2005) Chronopharmacological study of antidepressants in forced swimming test of mice. J Pharmacol Exp Ther 315(2):764–770 15. Dhir A, Kulkarni SK (2008) Involvement of sigma (r1) receptors in modulating the anti-depressant effect of neurosteroids (dehydroepiandrosterone or pregnenolone) in mouse tail-suspension test. J Psychopharmacol 22(6):691–696 16. Bjorklund A, Ehinger B, Falck B (1972) Analysis of fluorescence excitation peak ratios for the cellular identification of noradrenaline, dopamine or their mixtures. J Histo- chem Cytochem 20(1):56–64 17. Keith IM, Will JA (1981) Hypoxia and the neonatal rabbit lung: neuroendocrine cell numbers, 5-HT fluorescence intensity, and the relationship to arterial thickness. Thorax 36(10):767–773 18. Porsolt RD, Brossard G, Hautbois C et al. (2001) Rodent models of depression: forced swimming and tail suspension behavioral despair tests in rats and mice. Curr Protoc Neurosci. Chapter 8:Unit 8. 10A 19. Mu J, Xie P, Yang ZS, Yang DL et al (2007) Neurogenesis and major depression: implications from proteomic analyses of hippocampal proteins in a rat depression model. Neurosci Lett 416(3):252–256 20. Kawamoto K, Aoki J, Tanaka A et al (2002) Nerve growth factor activates mast cells through the collaborative interaction with lysophosphatidylserine expressed on the membrane surface of activated platelets. J Immunol 168(12):6412–6419 21. Cordeiro ML, Gundersen CB, Umbach JA (2004) Convergent effects of lithium and valproate on the expression of proteins associated with large dense core vesicles in NGF-differentiated PC12 cells. Neuropsychopharmacology 29(1):39–44 22. Zilkha-Falb R, Ziv I, Nardi N et al (1997) Monoamine-induced apoptotic neuronal cell death. Cell Mol Neurobiol 17(1):101– 118 23. Feng N, Hoover DB, Paolocci N (2007) Forever young?: nerve growth factor, sympathetic fibers, and right ventricle pressure overload. Circ Res 100(12):1670–1672 24. Pereira AC, Huddleston DE, Brickman AM et al (2007) An in vivo correlate of exercise-induced neurogenesis in the adult dentate gyrus. Proc Natl Acad Sci USA 104(13):5638–5643
123
1314 25. Santarelli L, Saxe M, Gross C et al (2003) Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 301:805–809 26. Jiang W, Zhang Y, Xiao L et al (2005) Cannabinoids promote embryonic and adult hippocampus neurogenesis and produce anxiolytic- and antidepressant-like effects. J Clin Invest 115:3104–3116 27. Lo CM, Ma L, Zhang DM, Lee R et al (2007) Mechanism of the induction of brain c-Fos -positive neurons by lipid absorption. Am J Physiol Regul Integr Comp Physiol 292:R268–R273 28. Zittel TT, Glatzle J, Kreis ME et al (1999) C-fos protein expression in the nucleus of the solitary tract correlates with cholecystokinin dose injected and food intake in rats. Brain Res 846(1):1–11
123
Neurochem Res (2010) 35:1302–1314 29. Emond M, Schwartz GJ, Moran TH (2001) Meal-related stimuli differentially induce c-Fos activation in the nucleus of the solitary tract. Am J Physiol Regul Integr Comp Physiol 280(5):R1315– R1321 30. Louneva N, Cohen JW, Han LY et al (2008) Caspase-3 is enriched in postsynaptic densities and increased in Alzheimer’s disease. Am J Pathol 173(5):1488–1495 31. Lirk P, Haller I, Hausott B et al (2006) The neurotoxic effects of amitriptyline are mediated by apoptosis and are effectively blocked by inhibition of caspase activity. Anesth Analg 102:1728–1733
