Cornel iridoid glycoside improves memory ability and promotes neuronal survival in fimbria–fornix transected rats

European Journal of Pharmacology 647 (2010) 68–74

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Neuropharmacology and Analgesia

Cornel iridoid glycoside improves memory ability and promotes neuronal survival in fimbria–fornix transected rats Li-hong Zhao a,b,1, Yue-xia Ding a,c,1, Lan Zhang a, Lin Li a,⁎ a b c

Department of Pharmacology, Xuanwu Hospital of Capital Medical University, Key Laboratory for Neurodegenerative Diseases of Ministry of Education, Beijing 100053, PR China Central Laboratory, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100026, PR China Department of Pharmacy, Yantai Yuhuangding Hospital, Yantai, Shangdong 264000, PR China

a r t i c l e

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Article history: Received 12 April 2010 Received in revised form 1 July 2010 Accepted 21 August 2010 Available online 6 September 2010 Keywords: Iridoid glycoside Cornus officinalis Hippocampus Fimbria–fornix transection Learning and memory Neurotrophin Synapses Apoptosis Alzheimer's disease Brain injury

a b s t r a c t Cornel iridoid glycoside (CIG) is a main component extracted from a traditional Chinese herb Cornus officinalis. Our previous study found that CIG improved neurological function in cerebral ischemic rats. The aim of this study was to investigate the therapeutic benefit of CIG in rats with fimbria–fornix transection (FFT) and explore the underlying molecular mechanisms. CIG (20, 60 and 180 mg/kg) or vehicle was intragastrically administered once daily to rats, starting immediately after the surgery and lasting for 4 weeks. Morris water maze and step-through tests showed that the memory deficits seen in FFT rats were significantly improved by CIG treatment. Immunohistochemical analysis showed that CIG treatment attenuated the loss of neurons in hippocampus. To elucidate the memory-improving mechanism of CIG, the neurotrophic factors, synaptic proteins and Bcl-2 family proteins in hippocampus were measured by Western blot analysis. FFT reduced hippocampal protein levels of nerve growth factor (NGF), tyrosine receptor kinase A (Trk A), brain-derived neurotrophic factor (BDNF), synaptophysin (SYP) and B-cell lymphoma-2 (Bcl-2), but not levels of tyrosine receptor kinase B (Trk B) and growth-associated protein 43 (GAP-43). FFT also elevated cytochorome C (Cyt c) and bcl-2-associated X protein (Bax). Administration of CIG to FFT rats significantly elevated the expression of NGF, TrkA, BDNF, SYP, GAP-43 and Bcl-2, and decreased the expression of Cyt c and Bax. These results indicated that CIG effectively counteracted cognitive impairments caused by fimbria–fornix lesions, and the mechanisms might be related to promoting neuronal survival and providing a beneficial environment for brain repair. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Cornus officinalis is a member of the Cornaceac family. This herb was first recorded in Shen Nong's Materia Medica about 2000 years ago in China, and is often used to tonify the liver and the kidney according to the theory of traditional Chinese medicine. In recent years, it has been found that some extracts from Fructus Corni, the sarcocarp of C. officinalis, exhibit a number of biological activities, including immunological regulation, blood glucose reduction, antishock, anti-arrhythmia, anti-inflammation and so on (Chinese Pharmacopoeia Commission, 2005). Cornel iridoid glycoside (CIG) is a main component extracted from Fructus Corni. Previous studies in our laboratory found that intragastrical administration of CIG improved neurological function, decreased cerebral infarct size, reduced nitric oxide and inhibited nuclear factor kappa B (NF-κB) expression in cerebral ischemic rats (Li et al., 2005; Zhang et al., 2007). We also found that CIG improved neurobehavioral outcomes and ⁎ Corresponding author. Tel.: + 86 10 8319 8886; fax: + 86 10 6304 2809. E-mail address: [email protected] (L. Li). 1 The first two authors contributed equally to this work. 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.08.016

promoted neurogenesis and angiogenesis 7, 14 and 28 days following focal cerebral ischemia induced by middle cerebral artery occlusion in rats (Yao et al., 2009). It therefore seems that the administration of CIG is able to exert neuroprotective and neurotrophic effects in cases of vascular damage to the brain. However, it is unknown whether CIG has the similar effects in cases of nonvascular damage to the brain. Bilateral fimbria–fornix transected (FFT) rats have served as the model of Alzheimer's disease or mechanical brain injury in recent years. Alzheimer's disease is an irreversible progressive disorder in which brain neurons deteriorate, resulting in the loss of cognitive functions, primary memory, judgment, reasoning, movement coordination and pattern recognition (Karlawish et al., 2005; Zarranz, 2004). The hippocampus has been implicated in both spatial learningmemory and passive avoidance memory. The fimbria–fornix constitutes a major afferent and efferent fiber tract connecting the hippocampus with the diencephalon, forebrain, striatum and prefrontal cortex (Cassel et al., 1997). Transections of the fimbria–fornix deprive the hippocampus of its major cholinergic input and, furthermore, disrupt substantial parts of the output from the hippocampal formation. Bilateral fimbria–fornix lesioning in rodents induces profound impairments of spatial learning and memory

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capabilities persisting for 6 months and is a model that reproduces some features of the cholinergic deficits and cognitive impairments found in Alzheimer's disease (Cassel et al., 1998; Krugel et al., 2001; Terry and Buccafusco, 2003) and in traumatic brain injury (Mala et al., 2008). Based on our previous studies on cerebral ischemia, we hypothesized that CIG might also exert neuroprotective and neurotrophic effects in cases of nonvascular damage to the brain. To test this hypothesis, we assessed the effects of CIG on the memory deficit induced by bilateral transection of the fimbria–fornix in the present study, and investigated the mechanisms by which CIG affected hippocampal neuron activities, including neurotrophic factors, synaptic proteins and apoptosis-related proteins. 2. Materials and methods 2.1. Subjects A total of 58 adult male Sprague–Dawley rats weighing 250–300 g were used after one week adaptation period (20 to 23 °C; 12 h light cycle; specific pathogen-free conditions; food and water ad libitum). The rats were randomly divided into five experimental groups: (1) Sham: sham operation group (n = 10); (2) Vehicle: vehicle (H2O)treated bilateral FFT model group (n = 12); (3) CIG-20: 20 mg/kg CIGtreated FFT group (n = 12); (4) CIG-60: 60 mg/kg CIG-treated FFT group (n = 12); (5) CIG-180: 180 mg/kg CIG-treated FFT group (n = 12). CIG or vehicle was intragastrically administered daily for 4 weeks starting 3 h after the surgery. All experiments followed the requirements of the Provisions and General Recommendations of Chinese Experimental Animal Administration Legislation. The suffering and the number of animals were minimized in all experimental conditions. 2.2. Fimbria–fornix transection surgery Rats underwent bilateral fimbria–fornix transection (FFT; n = 48) or sham operation (n = 10). All surgeries were performed under aseptic conditions in a sterile operating room. Rats were anesthetized with 10% chloral hydrate (350 mg/kg, i.p.). Bilateral FFT was performed using a wire knife attached to a stereotaxic frame (SN-2 type, Narishige Ltd, Japan). The skull was exposed and opened with a dental drill at stereotaxic coordinates 1.6 mm posterior to bregma and 1.2 mm lateral to midline (Paxinos and Watson, 1986). The wire knife was quickly lowered into the brain 5.5 mm ventral to dura at each lateral coordinate and kept for 2 min. Then the knife was slowly moved up 3 mm, to 2.5 mm ventral from dura. And the knife was lowered again and repositioned at 5.5 mm ventral to dura; the knife was moved up 3 mm, and the procedure repeated three times, to insure a complete cut through the fibers of the fornix. Identical procedures were performed in both hemispheres. Upon completion of surgery, the exposed skull was covered with sterile acrylic resin, and the incision was closed and treated with betadine to prevent infection. Sham surgery animals were operated by opening the skull only. The rats were placed on a heating pad during recovery from anesthesia to maintain the body temperature at 37.0 ± 0.5 °C after surgery. The efficiency of the axotomy was proven by Nissl's staining. 2.3. Cornel iridoid glycoside The sarcocarp of C. officinalis was purchased from the Tong-RenTang Company, Beijing, China. Cornel iridoid glycoside (CIG) was extracted from C. officinalis as described previously (Yao et al., 2009). The purity of CIG was 70% as determined by HPLC. CIG was dissolved in sterile distilled water at a concentration of 2, 6 or 18 mg/ml. Fresh solutions were prepared every 2 days. Equal volumes of CIG or vehicle according to animal weight (viz, 10 ml/kg of

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body weight) were intragastrically administrated at 8:00 to 9:00 AM and 60 min prior to each daily behavioral testing. 2.4. Cognitive measurements 2.4.1. Morris water maze test Following 21-day CIG or vehicle administration, cognitive measurements began. A spatial memory test was performed by the method of Morris et al. (1982) with minor modification as described in our previous reports (Wang et al., 2007; Wei et al., 2005; Zhang et al., 2006). This process of Morris water maze consisted of 5 days learning-memory training and a probe trial that was applied on day 6. On each day, rats were trained for one morning and one afternoon block. Each block consisted of two trials, and each trial lasted for 120 s, or ended if the rats reached the submerged platform. The escape latencies from the water maze (finding the submerged escape platform) were collected. The probe trial was formed by removing the platform and allowing each rat to swim freely for 60 s inside the pool. The swimming time and distance for each rat spent in the target quadrant (where the platform was removed) were recorded by a computerized video system. The ratio of time and distance in the quadrant to those in the whole pool was calculated to make up the comparisons between the groups. During all behavioral procedures the experimenter was kept ignorant about the group to which an individual rat belonged. 2.4.2. Step-through test The step-through test began at the day after Morris water maze test finished. In the present study, a two-compartment step-through apparatus was used to measure passive avoidance memory performance of rats. The box was divided into two identical light and dark compartments (20 × 20 × 20 cm) by a small door (5 × 5 cm). The bright compartment was illuminated by a white glow light and the floor of the non-illuminated dark compartment was composed of 2mm stainless steel rods spaced 1 cm apart. For the acquisition trial, rats were initially placed in the light chamber. When the rat's hind legs entered the dark chamber, an electrical foot shock (0.5 mA) lasting 3 s was delivered through the stainless steel rods. Twenty-four hours after the acquisition trial, the rats were again placed in the bright compartment for retention trials. The time taken for a rat before entering the dark compartment was measured as the latency in both acquisition and retention trials, with a maximum of 300 s. 2.5. Nissl staining After completion of behavioral testing, all animals were anesthetized with 10% chloral hydrate (4 ml/kg, i.p.) and transcardially perfused with saline followed by cold 4% paraformaldehyde in PBS. The brain was removed and equilibrated in a cryoprotectant solution of 30% sucrose/PBS and stored at 4 °C. The cryostat sections at 30 μm thickness were prepared and placed on microscope slides. Sections were Nissl stained according to standard methods (Parent et al., 2002). And the locus as well as size of lesions was verified. 2.6. Western blotting After completion of behavioral testing, rats were anesthetized and sacrificed, and the brains were quickly removed. Hippocampal tissues were dissected out, minced on ice, and then ice-cold lysis buffer (0.15 M NaCl, 1% TritonX100, 1% Deoxycholic acid sodium salt, 0.1% SDS, 10 mM Tris–HCl pH 7.4, 1% phenylmethylsulfonylfluoride). Brain lysates were prepared in sodium dodecylsulfate (SDS)-containing sample buffer and 20 μg protein was loaded into each lane of 12% SDSpolyacrylamide gels. After transferring to a PVDF membrane (Hybond, America), blots were probed with antibodies to nerve growth factor (NGF; 1:2000 dilution; Abcam), Trk A (1:1000; Chemicon), brain-

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derived neurotrophic factor (BDNF; 1:1000; Santa Cruz), Trk B (1:1000; Santa Cruz), growth-associated protein-43 (GAP-43; 1:1000; Santa Cruz), synaptophysin (SYP; 1:1000; Santa Cruz), Bax (1:500; Santa Cruz), Bcl-2 (1:500; Santa Cruz), and cytochrome c (1:500; Santa Cruz), respectively. Membranes were then incubated with appropriate secondary antibodies (1:2000 dilution), followed by chemiluminescence detection (NEN Life Science Products, Boston, MA), and then exposed to Kodak Biomax film (Eastman Kodak, Rochester, NY). Images were captured through a Charge Coupled Device camera, and gel bands were analyzed using the GIS 1D Gel Image System ver. 3.73 (Tanon, Shanghai, China). Raw data were converted to relative values, and these relative values were expressed as percent of the vehicle-treated FFT average. To assess consistency of loading, each blot was reblotted with an antibody against β-actin.

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2.7. Statistical analysis

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3. Results 3.1. FFT model validation by anatomical site Nissl-stained sections were examined 28 days after FFT operation in rats. Based on histological verification, data from 1 lesioned animal from the Vehicle group, 2 lesioned rats in the CIG-20 group and 1 lesioned animal in the CIG-180 group were discarded due to unilateral or poorly placed lesions. Thus data from a total of 54 animals were used in the analyses. Histological analysis of the remaining animals revealed complete bilateral transection of fimbria–fornix fibers, as illustrated in Fig. 1 from a representative animal. 3.2. Effects of CIG on learning and memory impairment in FFT rats 3.2.1. Morris water maze test Morris water maze test was used to evaluate the spatial learning and memory ability in rats 21 days after FFT operation. Significant group differences in escape latencies to platform are illustrated in Fig. 2A. The escape latencies were at similar high level on the first day training in all groups. During the period from Day 2 to Day 5, the escape latencies were decreased and remained at a low level in the sham-operated control rats; FFT increased the escape latencies compared with sham group; intragastrical administration of CIG (20, 60 and 180 mg/kg) decreased the escape latencies compared with vehicle-treated model group.

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All data were expressed as mean± standard error (mean± S.E.M.). Morris water maze latencies were analyzed by SPSS 10.0 using Windows software to conduct repeated measures ANOVA. Other data were analyzed by one-way ANOVA (equal variances assumed by S-N-K). The data were considered to be statistically significant if the probability had a value of 0.05 or less.

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Fig. 2. Effect of cornel iridoid glycoside (CIG) on spatial learning and memory (Morris water maze task) in FFT rats. CIG (20, 60 and 180 mg/kg) or vehicle was administered i.g. once a day for 28 days, which lasted up to the day of sacrifice. (A) Comparison of escape latencies to find the platform during five training days. Each rat was subjected to four trials per day. (B) Comparison of the percentage of searching time and distance spent in the target quadrant where the platform was removed in probe trial. The data from each group are summarized by the mean ± S.E.M. (n = 10–12). ##: P b 0.01, significantly different from the sham-operated control rats. *: P b 0.05, **: P b 0.01, significantly different from the vehicle-treated FFT model rats.

Fig. 2B illustrates the group differences in the percentage of the searching time and distance in the target quadrant to whole pool during the probe trial. Bilateral FFT caused a decrease in the searching time and distance compared with sham operation group (P b 0.01); the treatment with CIG at doses of 20, 60 and 180 mg/kg showed significant longer searching time and distance in the target quadrant compared with vehicle-treated model group (P b 0.05, P b 0.01). These results indicated that CIG improved spatial learning and memory ability in FFT rats.

B

Fig. 1. Illustration of coronal Nissl-stained section through the fornix of a representative lesioned animal. (A) The intact fornix of sham control rat; (B) the fornix of fimbria– fornix transected (FFT) rat. The magnification was × 100.

3.2.2. Step-through test Step-through test was used to evaluate the passive avoidance memory in rats 28 days after FFT operation. Fig. 3 illustrates the performance of the five experimental groups. The latency in the acquisition training on the first day did not show obvious differences among five groups. In the retention testing 24 h later, FFT model rats exhibited a significant decrease in latency before entering the dark compartment (P b 0.01). Compared with vehicle-treated FFT group, rats treated with CIG at the doses of 60 and 180 mg/kg obviously prolonged the latency (P b 0.05), indicating that CIG improved the passive avoidance memory ability in FFT rats.

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Fig. 3. Effect of CIG on passive avoidance memory (step-through task) in FFT rats. CIG (20, 60 and 180 mg/kg, i.g.) or vehicle was administered for 28 days. The acquisition training on the first day and the retention testing 24 h later were carried out, and the latency was recorded. Data represent mean ± S.E.M. (n = 10–12). ##: P b 0.01, significantly different from the sham operation group. *: P b 0.05, significantly different from the vehicle-treated FFT model rats.

3.3. Effect of CIG on nerve cell injury in hippocampus of FFT rats

3.4. Effects of CIG on the expression of NGF, BDNF and their receptors in the hippocampus of FFT rats The expression of NGF and its receptor Trk A, and BDNF and its receptor Trk B in the hippocampus was detected by Western blotting (Fig. 5). Compared with sham operation group, the expression of NGF, Trk A and BDNF was decreased markedly in the hippocampus following FFT (P b 0.01). Compared with the vehicle-treated FFT model group, rats treated with CIG at the doses of 60 and 180 mg/ kg showed an increase in NGF expression (P b 0.01), and CIG at all three doses of 20, 60 and 180 mg/kg elevated the expression of TrkA and BDNF (P b 0.05, P b 0.01). However, Trk B level did not show significant differences among five groups. 3.5. Effects of CIG on the expression of synaptophysin and growthassociated protein-43 in the hippocampus of FFT rats Fig. 6 shows that the expression of synaptophysin (SYP) was decreased markedly in the hippocampus following FFT compared with sham operation controls (P b 0.01); rats treated with CIG at doses of 20 and 60 mg/kg demonstrated an increase in SYP level compared with vehicle-treated FFT model group (P b 0.05, P b 0.01). The level of growth-associated protein-43 (GAP-43) showed no significant change in the hippocampus after FFT compared with sham controls; but rats treated with CIG at all three doses of 20, 60 and 180 mg/kg had an increase in GAP-43 level compared with vehicle-treated model rats (P b 0.01). 3.6. Effects of CIG on the expression of Bcl-2 family proteins and cytochrome c in the hippocampus of FFT rats Fig. 7 illustrates the expression of Bcl-2, Bax and cytochrome c (Cyt c) by using Western blot analysis. The expression of Bcl-2 protein was

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Fig. 4 displays the photomicrographs of pyramidal cells in the hippocampal CA1 region and dentate gyrus by using Nissl's staining. FFT caused nerve cell injury, evidenced by thin granular layer, nucleus shrinkage or disappearance, and reduced density of Nissl's bodies when compared with sham operation group (P b 0.01). With the treatment of CIG (20, 60 and 180 mg/kg), the morphology of nerve cells was in tendency to normal and the density of Nissl's bodies was increased as compared with the vehicle-treated model rats (P b 0.05, P b 0.01).

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Fig. 4. Effect of CIG on pyramidal cell injury in the hippocampus of FFT rats (Nissl's staining). (A) Photomicrographs in the hippocampal CA1. (B) Photomicrographs in the dentate gyrus subregion. (C) Image analysis of positive pixel count of Nissl's staining. The data are from 15 sections of each group (three sections per rat) and expressed as mean ± S.E.M. ##: P b 0.01, significantly different from the sham operation group.*: P b 0.05, **: P b 0.01, significantly different from the vehicle-treated FFT model rats. Scale bar = 100 μm.

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Fig. 5. Effects of CIG on protein expression of NGF, Trk A, BDNF and Trk B in the hippocampus of FFT rats. (A) Western blot bands of NGF, Trk A, BDNF and Trk B. β-actin was performed to standardize the expression of proteins. (B) Quantitative analysis of relative changes in the expression of NGF, Trk A, BDNF and Trk B. Data are expressed as mean ± S.E.M. of percentage of animals in each group to vehicle-treated FFT rats. n = 3 (the data of each group were obtained from three rats in three experiments). ##: P b 0.01, significantly different from the sham operation group. *: P b 0.05, **: P b 0.01, significantly different from the vehicle-treated FFT model rats.

decreased markedly in hippocampus following FFT compared with sham controls (P b 0.01); rats treated with CIG at all three doses showed an increase in Bcl-2 level compared with vehicle-treated model group (P b 0.05, P b 0.01). Meanwhile, the levels of Bax protein and Cyt c showed a significant increase after FFT compared with sham controls (P b 0.01). Treatment with CIG at the doses of 20 and 60 mg/ kg decreased Bax expression (P b 0.05, P b 0.01), and CIG at the doses of 60 and 180 mg/kg declined Cyt c level when compared with vehicletreated model group (P b 0.05). 4. Discussion We previously revealed that CIG improved neurological function in case of cerebral ischemia (Yao et al., 2009). In the present study we expand these results by demonstrating that CIG was able to alleviate the cognitive/behavioral impairment of nonvascular brain injury in the form of bilateral fimbria–fornix transacted (FFT) rats. Impaired performances of learning and memory tasks in FFT animals include

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60 180 FFT/CIG (mg/kg)

Fig. 6. Effects of CIG on the expression of synaptophysin (SYP) and growth-associated protein-43 (GAP-43) in the hippocampus of FFT rats. (A) Western blot bands of SYP and GAP-43. β-actin was performed to standardize the expression of proteins; (B) Quantitative analysis of relative changes in the expression of SYP and GAP-43. Data are expressed as mean ± S.E.M. of percentage of animals in each group to vehicle-treated FFT rats. n = 3 (the data of each group were obtained from three rats in three experiments). ##: P b 0.01, significantly different from the sham operation group. *: P b 0.05, **: P b 0.01, significantly different from the vehicle-treated FFT model rats.

deficits in spatial reference memory (Cassel et al., 1998; Morris et al., 1982) and in passive avoidance learning (Deacon et al., 2002). In our present study, the vehicle-treated FFT rats also showed substantial impairment of task acquisition, including Morris water maze test for the spatial learning-memory and step-through test for the passive avoidance memory. The groups subjected to similar lesions but treated with CIG, on the other hand, were less severely impaired — performing at a level significantly superior to that of the similarly lesioned vehicle-treated group (Figs. 2 and 3). Consequently, these groups demonstrate that administration of CIG immediately after lesions of the FF is able to reduce the lesion-associated impairment of cognitive function. After the behavioral measurement, we have investigated the mechanisms by which CIG improves learning and memory abilities in FFT rats. Transection of FF may induce a decrease in hippocampal pyramidal cells and dentate gyrus granule cells (He et al., 1992; Krugel et al., 2001). The loss of septal and hippocampal cholinergic and noncholinergic neurons after FFT can be assessed by measurement of Nissl-stained cell bodies (Naumann et al., 1994; Naumann et al., 1992). Nissl's body, a normal intracytoplasmic structure of neuron, is distributed in cell body and large dendrites, and consists of many rough endoplasmic reticulums and ribosomes. It is an apparatus for protein synthesis, and can serve as an indicator to observe the functional status of nerve cells. From the experimental results of the present study, we found the reduced amount of Nissl-stained cell bodies, and the thinned pyramidal layer in the hippocampal CA1

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A Bcl-2

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Fig. 7. Effects of CIG on protein expression of Bcl-2, Bax and cytochorome c in the hippocampus of FFT rats. (A) Western blot bands of Bcl-2, Bax and cytochorome c (Cyt c). β-actin was performed to standardize the expression of proteins. (B) Quantitative analysis of relative changes in levels of Bcl-2, Bax and Cyt c. Data are expressed as mean± S.E.M. of percentage of animals in each group to vehicle-treated FFT rats. n = 3 (the data of each group were obtained from three rats in three experiments). ##: P b 0.01, significantly different from the sham operation group. *: P b 0.05, **: P b 0.01, significantly different from the vehicle-treated FFT model rats.

region and granular layer in the dentate gyrus of FFT rats. The treatment with CIG to FFT rats increases the number of Nissl-stained nerve cells, and thickens pyramidal layer and granular layer (Fig. 4). It is suggested that decreasing the loss of neurons in hippocampus by CIG may contribute to its improving cognitive impairment in FFT rats. To elucidate why CIG decreases the cell loss and/or promotes nerve growth, we have further investigated CIG's mechanisms in three aspects: neurotrophic factors, synaptic proteins and apoptosis-related proteins. The family of neurotrophins consists of the proteins NGF, BDNF, neurotrophin-3 (NT-3), NT-4/5, and NT-6. Neurotrophins exert their cellular effects by interaction with their receptors. The Trk receptor family consists of three tyrosine specific receptor kinases (TrkA, TrkB and TrkC) showing ligand selectivity. TrkA has been identified as the preferred receptor for NGF, TrkB for both BDNF and NT-4/5, and TrkC for NT-3. Neurotrophins are initially involved in the embryogenesis and organogenesis. They regulate both synaptic activity and neurotransmitter synthesis and they control neural plasticity in adults (Schulte-Herbruggen et al., 2007). Previous studies in our laboratory found that after ischemia CIG improved neurological function and promoted neurogenesis (Yao et al., 2009). The local microenvironment in regions of ongoing neurogenesis in the adult mammalian

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brain regulates the self-renewal, activation and differentiation of stem cells (Spradling et al., 2001; Wurmser et al., 2004). Neurotrophic factors play a positive role in microenvironment. Among the neurotrophins, BDNF is known for its survival-promoting effects on new neuroblasts through its receptor TrkB (Bath et al., 2008). NGF also enhances survival of new hippocampal neurons, most likely by increasing cholinergic tone (Frielingsdorf et al., 2007). For Alzheimer's disease treatment, research has reached the point of creating strategies for therapeutic intervention with neurotrophins. NGF can protect axotomized septal cholinergic neurons from the atrophy and death otherwise induced by fimbria–fornix lesions (Hefti et al., 1989). This finding, along with other observations demonstrating a role for retrograde NGF-transport from hippocampus to septum in the maintenance of basal forebrain cholinergic neurons (Capsoni and Cattaneo, 2006; Salehi et al., 2006), has led to several clinical trials using NGF for Alzheimer's disease. However, in human brain the administration of neurotrophin induces enormous adverse reaction as pain and weight loss (Schulte-Herbruggen et al., 2007) limiting its possible therapeutic utilization. Thus CIG may offer the alternative strategies to boost endogenous protective molecules, for example NGF, BDNF content and/or its receptor activity in relevant brain areas while preventing the onset of adverse effects. In the present study, reduction of NGF protein and its Trk A receptor in the hippocampus caused by lesion of the FF are attenuated by treatment with CIG. As to BDNF and its Trk B receptor, though there is no change of Trk B in the hippocampus after FFT, CIG treatment also cause elevations of BDNF (Fig. 5). Thus a CIG treatment-related increase of the cerebral NGF, Trk A and BDNF may prevent neuronal loss and contribute to the cognition improvement. Neurotrophic factors offer the additional potential benefit that they influence not only the survival of target neurons, but also their synaptic functions (Hempstead, 2006; Schulte-Herbruggen et al., 2007). Synaptic loss resulting in local and global disconnection within the telencephalon may account for cognitive deficits that occur at earlier stages of chronic neurodegenerative disorders (Terry, 2000). A consistent observation in studies of Alzheimer's disease brains in comparison to non-Alzheimer diseased controls is the loss of synapses, which is also correlated with dementia severity (Scheff and Price, 2003; Scheff et al., 2006). Synaptophysin (SYP) is the most established and widely used synaptic protein, being present in virtually all synaptic terminals and usually taken to be a marker of synaptic distribution and synaptic density (Gincel and Shoshan-Barmatz, 2002). It takes part in the development of nerve synapses and adjusts plasticity of nerve synapses (Tarsa and Goda, 2002). The present study demonstrates that SYP protein expression in the hippocampus exhibits a decreasing trend in the FFT group after the injury. In contrast, in the CIG-treated groups, significant increases of SYP are observed (Fig. 6). These results suggest that SYP might represent a potential presynaptic molecular substrate by which CIG increases hippocampal synaptic plasticity. Growth-associated protein-43 (GAP-43) is another presynaptic protein which is considered to be a molecular marker of neuron growth and plasticity (Carulli et al., 2004). Our results show that GAP-43 protein expression remained unchanged in hippocampus after FFT, but CIG treatment enhances the expression of this protein in FFT rats (Fig. 6). Thus, the data show that CIG may maintain the capacity of plasticity by increasing SYP and GAP-43 levels in the hippocampus to alleviate the loss of neuron plasticity in response to damage and overcome some injury-related deficiencies in neuronal function. Our previous studies indicated that CIG decreased NF-κB expression, and regulated apoptosis-related factors in ischemic rats (Li et al., 2005). Therefore, we presume that CIG may alleviate the loss of neurons by inhibiting apoptosis in nonvascular injury to brain. Apoptosis is an active energy-dependent mode of cell death and is regulated by tightly controlled intracellular signaling events

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(Schwartzman and Cidlowski, 1993). The inappropriate activation of apoptosis may contribute to a variety of pathogenic processes such as the loss of neuronal cells in Alzheimer's disease, stroke and traumatic brain injury. Members of the Bcl-2 family proteins are key regulators of apoptosis by acting on the mitochondria (Green and Reed, 1998). This protein family includes both anti-apoptotic molecules such as Bcl-2 and Bcl-X (L), and pro-apoptotic molecules such as Bax, Bak, Bid, and Bad. Many other studies have demonstrated that Bcl-2 family proteins are related to the formation of channels in mitochondrial membranes and regulate cytochrome c (Cyt c) release. The released Cyt c from mitochondria may activate the intrinsic apoptotic pathway via apoptosome formation and caspase-9 activation and thus drive cells to apoptosis (Kluck et al., 1997; Yang et al., 1997). In the present experiment, we found downregulation of Bcl-2 and upregulation of hippocampal Bax and Cyt c protein in rats suffered from FFT. And CIG treatment counteracted the effects of FFT on Bcl-2, Bax and Cyt c (Fig. 7). These results suggest that CIG may suppress apoptosis induced by FFT. Thus the regulatory effects of CIG on the Bcl-2 family proteins might contribute to a beneficial microenvironment for nerve growth and repair after brain injury. In conclusion, the overall evidence in the present study indicates that CIG treatment alleviates the cognitive/behavioral impairment of FFT, and the mechanisms might be related to CIG's promoting neuronal survival and growth, increasing the levels of neurotrophins, improving synaptic functions and suppressing apoptosis in the hippocampus. The neural and cognitive mechanisms by which CIG exerts its symptom-reducing and/or recovery-promoting effects after FFT are still far from being fully understood. We do, however, by now have promising indications that intragastrically administered CIG is able to exert therapeutic effects in cases of nonvascular damage to the brain, which will be beneficial to the future utilization of this therapeutic choice in the treatment of Alzheimer's disease patients or traumatic brain injury. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 90709011, 30973513, and 30701092); Beijing Science and Technology Program (No. D0206001043191). We would like to thank Cui-fei Ye and Li Zhang for technical assistance and Dr. Lin-lin Yin for her helpful suggestions on the manuscript. References Bath, K.G., Mandairon, N., Jing, D., Rajagopal, R., Kapoor, R., Chen, Z.Y., Khan, T., Proenca, C.C., Kraemer, R., Cleland, T.A., Hempstead, B.L., Chao, M.V., Lee, F.S., 2008. Variant brain-derived neurotrophic factor (Val66Met) alters adult olfactory bulb neurogenesis and spontaneous olfactory discrimination. J. Neurosci. 28, 2383–2393. Capsoni, S., Cattaneo, A., 2006. On the molecular basis linking Nerve Growth Factor (NGF) to Alzheimer's disease. Cell. Mol. Neurobiol. 26, 619–633. Carulli, D., Buffo, A., Strata, P., 2004. Reparative mechanisms in the cerebellar cortex. Prog. Neurobiol. 72, 373–398. Cassel, J.C., Duconseille, E., Jeltsch, H., Will, B., 1997. The fimbria–fornix/cingular bundle pathways: a review of neurochemical and behavioural approaches using lesions and transplantation techniques. Prog. Neurobiol. 51, 663–716. Cassel, J.C., Cassel, S., Galani, R., Kelche, C., Will, B., Jarrard, L., 1998. Fimbria–fornix vs selective hippocampal lesions in rats: effects on locomotor activity and spatial learning and memory. Neurobiol. Learn. Mem. 69, 22–45. Chinese Pharmacopoeia Commission, 2005. Pharmacopoeia of People's Republic of China. Chemical Industry Press, Beijing, p. 20. Deacon, R.M., Bannerman, D.M., Kirby, B.P., Croucher, A., Rawlins, J.N., 2002. Effects of cytotoxic hippocampal lesions in mice on a cognitive test battery. Behav. Brain Res. 133, 57–68.

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