OL Oncology Letters 1792-1074 1792-1082 D.A. Spandidos 10.3892/ol.2017.7333 OL-0-0-7333 Articles Expression of nerve growth factor and brain-derived neurotrophic factor in astrocytomas LiuTing-Ting 1 2 * WangHao 3 * WangFeng Juan 2 XiYu Feng 4 ChenLi Hua 2 Department of Internal Medicine, No. 4 West China Teaching Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China Department of Hematology and Research Laboratory of Hematology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R. China Department of Gastrointestinal Surgery, The First Affiliated Hospital of Chengdu Medical College, Chengdu Medical College, Chengdu, Sichuan 610041, P.R. China State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, P.R. China Correspondence to: Dr Hao Wang, Department of Gastrointestinal Surgery, The First Affiliated Hospital of Chengdu Medical College, Chengdu Medical College, 4 Baoguang Road, Xindu, Chengdu, Sichuan 610041, P.R. China, E-mail: hw0616@sina.com

Contributed equally

 01 2018 02 11 2017 15 1 533 537 30062016 18052017 Copyright © 2018, Spandidos Publications 2018

Neurotrophic factors (NTFs) are well known to serve critical functions in neural survival, neurite growth and cell differentiation in vivo and in vitro. Previous progress has indicated that nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), two NTF family members, may be involved in the process of tumor progression. In the present study, the expression of NGF and BDNF was detected using immunohistochemistry on 70 adult astrocytoma samples collected from distinct locations as well as of various pathological grades, with an additional 15 samples being collected from normal adult brain tissue to be used as controls. NGF and BDNF were identified to be expressed in all samples, and their positive cell expression rates in astrocytomas demonstrated a significant increase compared with that in the normal controls (P<0.05), particularly in grade III (P<0.05). In addition, the expression of NGF and BDNF exhibited a gradual decrease ranging from the temporal lobe, parietal lobe and cerebellum to the frontal lobe (P<0.05). The results of the present study suggest that the expression of NGF and BDNF is increased in astrocytomas, which is associated with the pathological grade and the astrocytoma location.

 nerve growth factor brain-derived neurotrophic factor astrocytoma Introduction

Neurotrophic factors (NTFs) are endogenously produced as biological mediators that regulate neural growth, differentiation and apoptosis in the development of the nervous system (1–3). Nerve growth factor (NGF) is the most well-known and best characterized member of the neurotrophin family (1,4,5), which is structurally similar to other growth factors including brain-derived neurotrophic factor (BDNF) (1,6,7). NGF is produced by cells located in several brain regions, and is expressed at the highest levels in the hippocampus, cortex (1,5,8), cerebellum and spinal cord (1,7,9). NGF, and receptors, tropomyosin receptor kinase A (TrkA) and p75 nerve growth factor receptors, exhibit pleiotropic effects in inducing cell differentiation, preventing apoptosis and supporting cell survival in neuronal progenitor cells and immature neurons of the central nervous system (CNS) and peripheral nervous system (1,6,10,11). Previous studies have demonstrated that NGF and TrkA may contribute an important role in the growth modulation of human tumor cells (12). BDNF, the second member of the NTFs to be identified, is widely distributed in the CNS (13–17), and is produced primarily in the hippocampus, thalamus and cerebellum (1,18). BDNF serves an essential role in maintaining and modulating the physiological functions of neurons and promoting the growth, survival and metastasis of a variety of tumor cells through binding to its receptors, tropomyosin receptor kinase B (TrkB) and p75 nerve growth factor receptors (19–24).

Previous studies have suggested that NTFs and their receptors are associated with cell maturation and tumoral cell apoptosis (1,25,26). In contrast, a number of growth factors and signaling molecules appear to represent specific markers for distinct histological types of tumors and may be involved in the differentiation process of neoplasms (1). In accordance with these distinct mechanisms of action of NTFs, the aim of the present study was to examine the expression of NGF and BDNF in astrocytomas, and to investigate the association between NGF, BDNF, pathological grading and location, as well as tumorigenesis of astrocytomas.

 Materials and methods <sec> <title>Tissue samples

A total of 70 clinical astrocytoma samples (39 male and 31 female, age range 21–75 years; median age 47.3 years) were collected from The First Affiliated Hospital of Chengdu Medical College (Chengdu, China) from February 2006 to May 2013. All procedures were performed according to an Institutional Review Board (IRB)-approved protocol. These samples, based on their pathological grades, were classified as 10 cases of grade I, 24 of grade II, 26 of grade III and 10 of grade IV. Conversely, grouped according to the location of astrocytomas, there were 20 samples from the temporal lobe, 16 from the frontal lobe, 18 from the parietal lobe and 16 from the cerebellum. In addition, 15 normal brain specimens were obtained as controls from the normal brain tissues (7 samples from the temporal lobe, 6 from the frontal lobe, 1 from the parietal lobe and 1 from the cerebellum) of patients who underwent surgery for cerebral trauma. The expression of NGF and BDNF was examined in different locations in astrocytomas, not in normal brain tissues, because it is relatively difficult to obtain the samples of normal brain tissues from locations other than the temporal lobe and frontal lobe.

The samples were obtained from patients during surgery, fixed in 10% neutralized formalin for 12 h at room temperature within 24 h of being obtained and subsequently dehydrated in graded ethanol, embedded in paraffin and then sliced into serial 4 µm sections. Each specimen was cut into 3 sections, which were used for immunohistochemistry, stained with hematoxylin and eosin together with a negative control (PBS).

 Reagents

A rabbit anti-human NGF polyclonal antibody (cat. no. MFCD07370460; 1:200 dilution) and a rabbit anti-human BDNF polyclonal antibody (cat. no. SAB2108004; 1:200 dilution) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Streptavidin-Peroxidase kit (cat. no. kit-9710) and diaminobenzidine (DAB) chromogenic reagent kit (DAB-2031) were purchased from Maixin New Biology (Fuzhou, Fujian, China).

 Immunohistochemistry

The sections were deparaffinized in xylene, rehydrated using serial dilutions of ethanol and washed three times in 0.1 M PBS for 5 min each time. Antigen retrieval was performed for 15 min at 100°C in citrate buffer (10 mmol/l; pH 6.0) in a microwave oven. The sections were then washed three times in 0.1 M PBS for 5 min each, and incubated at room temperature in 0.2% hydrogen peroxide for 20 min to block the action of any endogenous peroxidase. Following a further three 5 min washes in 0.1 M PBS, normal goat serum was added for 20 min at 37°C prior to incubation overnight at 4°C in one of the primary antibody solutions (polyclonal neurotrophin antisera for NGF and BDNF). Subsequently, the sections were washed in 0.1 M PBS three times for 5 min each prior to incubation with labeled streptavidin-biotin for 30 min at room temperature according to the manufacturer's protocol in Maixin New Biology. Thereafter, they were washed in 0.1 M PBS again, and DAB chromogenic reagent was added in order to observe the distribution of NGF and BDNF according to the manufacturer's protocol (Maixin New Biology). Finally, all sections were mounted, dehydrated, placed on coverslips and observed under a light microscope at magnification, ×200.

The tumor specimens were used as the experimental group (grade I–IV groups) with the normal brain tissue used as the control group. Instead of using the primary antibody as a negative control, PBS was used for both experimental and normal groups.

 Microscopic examination

Each immunohistochemistry section was observed under a light microscope (XS-212; Nanjing Jiangnan Novel Optics, Co., Ltd., Nanjing, China). The number of positively stained cells was determined from 10 fields of view at magnification, ×400 and the mean positive cell numbers were calculated independently by two pathologists from the Department of Pathology at The First Affiliated Hospital of Chengdu Medical College. Subsequently, the total cell number in the same fields of view was determined and the mean number of cells for each section was calculated.

Expression rate of positive cells=(number of positive cells/number of total cells) ×100%.

 Statistical analysis

The data and the categorical variables were analyzed and compared using analysis of variance (ANOVA) followed by a Student-Newman-Keuls post-hoc test, using the SPSS software package for Windows (version 11.5; SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation and P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Expression of NGF and BDNF in normal brain tissues and in astrocytomas of distinct pathological grades

The immunopositive products of NGF (Fig. 1) and BDNF (Fig. 2) were observed in the normal brain tissues and in the astrocytomas. The positive expression rate of NGF in astrocytomas was significantly increased compared with that in normal brain tissue (P<0.05). Furthermore, there was a statistically significant difference (P<0.05) in the expression rate of NGF- and BDNF-positive cells between distinct pathological grades of astrocytomas, with the highest expression observed in grade III (Table I; P<0.05).

The staining of NGF-positive cells was observed in the cytoplasm and nucleus, but primarily in the nucleus (Fig. 1). However, the staining of BDNF-positive cells was primarily observed in the cytoplasm (Fig. 2).

 Expression of NGF and BDNF in astrocytomas from distinct locations

NGF and BDNF were expressed in astrocytomas from all locations; however, the expression rate of positive cells was statistically different (P<0.05) between distinct locations. The sources may be ranked in decreasing order of expression rate from the temporal lobe, parietal lobe and cerebellum to the frontal lobe (Table II; P<0.05).

 Discussion

NTFs serve a crucial role in a variety of distinct types of brain cell and in their differentiation processes (1,7–9). Previous studies have suggested that NTFs may be associated with certain biochemical and molecular mechanisms of carcinogenesis and growth signaling pathways, undergoing deregulation in numerous human malignancies, including neuroectodermal brain tumors, testicular germ cell tumors and prostate cancer (1,27–30). However, little has been known previously regarding the association between the expression of NGF and BDNF, pathological grading, and region of astrocytomas. The results of the present study demonstrated that NGF and BDNF levels were increased in astrocytomas compared with the controls, and were associated with the pathological grading and region of astrocytomas.

The results of the present study revealed that NGF and BDNF were overexpressed in astrocytomas and were associated with the location, generation, progression and pathological grade of the astrocytoma. Goustin et al (31) demonstrated that the amount of serum required in the process of tumor cell growth was decreased and there was a ubiquitous decreasing demand for growth factors from extrinsic sources in numerous types of cancer cell. Specifically, the decrease may be restored via activating the autocrine pathway, altering the synthesis of growth factor receptors and activating the post-receptor machinery (31). The results of the present study demonstrated that the expression of NGF and BDNF were significantly upregulated gradually from normal tissues to grade I, II and III astrocytomas. This suggested that an autocrine pathway may be activated in the process of astrocytoma tumorigenesis and the proliferation of cancer cells was accelerated by synthesizing autocrine growth factors. However, the expression of NGF and BDNF in grade IV astrocytomas decreased suddenly, which was significantly different from in grade I, II and III astrocytomas. This may have resulted from the tumor cells no longer requiring the activation of the NGF receptor pathway. Therefore, it is hypothesized that the overexpression of NGF and BDNF may be the early and middle events in astrocytoma tumorigenesis.

In normal brain, the in situ hybridization experiment verified that NGF mRNA is expressed in pyramidal cells, granular cells in the hippocampus, stellate cells, oligodendrocytes and certain cortical neurons. NGF is produced by cells located in a number of brain regions, and is expressed in the highest amounts in the hippocampus and cortex (5,8). BDNF is produced primarily in the hippocampus, thalamus and cerebellum (18). The results of the present study identified that the expression of NGF and BDNF exhibited a statistically significant gradual decrease in the temporal lobe near the hippocampus, parietal lobe near the cerebral cortex, cerebellum and frontal lobe respectively. This phenomenon suggested that hippocampal and cortical tissues are activated to synthesize and release NGF and BDNF in the formation of astrocytomas.

NGF and BDNF are able to promote the survival and differentiation of neurons and contribute a key role in the reparative process of injured nerve cells. Multiple previous studies have also demonstrated that NGF and BDNF and their receptors are associated with tumor biology, behavior and progression (1,29,32), although the underlying molecular mechanism of tumor progression remains unclear. Zhu et al (33) identified that NGF and TrkA were notably overexpressed in pancreatic cancer tissues compared with in normal pancreatic tissue, and increased NGF/TrkA expression was associated with the invasion and the pain caused by the tumor, although it was not associated with the degree of tumor differentiation or grading. Furthermore, NGF is notably downregulated in esophageal carcinoma tissues and is associated with the degree of tumor differentiation as well as its pathological grades. Low TrkA expression was associated with advanced tumor stage (34). In addition, NGF mRNA and protein are expressed in breast cancer, but not in normal breast tissue, and may be inhibited by NGF-neutralizing antibodies or a TrkA-blocking agent such as K-252a (35). A study by Breit et al (36) demonstrated that TrkA was associated with the differentiation, generation and angiogenesis of neuroblastoma (NB). Another study demonstrated that the TrkA gene is a good prognostic marker in NB and inhibited NB hyperplasia, induced benign differentiation and affected the growth of blood vessels of NB (37). NGF and TrkA were also observed to be decreased, whereas p75 was increased in childhood low-grade astrocytomas and ependymoma tissue (1). Similarly, BDNF and TrkB are expressed in multiple myeloma cell lines (38). BDNF was also identified to induce cell migration and cell proliferation in cultured human ovarian cancer cells (39). In addition, the level of BDNF in serum was significantly associated with tumor size in patients with hepatocellular carcinoma (19). These studies and the results of the present study revealed that NGF and BDNF, with receptors, participate in tumorigenesis.

The results of the present study indicate that NGF and BDNF overexpression in astrocytomas and their positive cell rate may be ranked in decreasing order from grade III, II, IV and I, and, finally, normal brain tissue. This suggests that NGF and BDNF are involved in the process of astrocytoma tumorigenesis, and the overexpression of NGF and BDNF may occur at early and middle stages of astrocytoma formation. The results of the present study also identified that the closer to the hippocampal and cortical areas, the higher the expression rate of positive cells for NGF and BDNF in astrocytomas. This may be that the astrocytoma stimulates astrocyte cells to secrete NGF and BDNF during the process of astrocytoma formation, and the hippocampal and cortical regions produce growth factors including NGF and BDNF.

In conclusion, the results of the present study suggest that NGF and BDNF are overexpressed in astrocytomas, with associations with the pathological grade as well as the location of the astrocytomas.

 Acknowledgements

The authors would like to thank Professor Yuan Chun Fan and Dr Liang Jiang, from the Department of Pathology at The First Affiliated Hospital of Chengdu Medical College (Chengdu, China) for their technical assistance and helpful discussion.

 References ChiarettiAAloeLAntonelliARuggieroAPiastraMRiccardiRTamburriniGDi RoccoCNeurotrophic factor expression in childhood low-grade astrocytomas and ependymomasChilds Nerv Syst20412419200410.1007/s00381-004-0959-615138791 AloeLTirassaPBracci-LaudieroLNerve growth factor in neurological and non-neurological diseases: Basic findings and emerging pharmacological prospectivesCurr Pharm Des7113123200110.2174/138161201339838311172703 DragoJKilpatrickTJKoblarSATalmanPSGrowth factor: Potential therapeutic applications in neurologyJ Neurol Neurosurg Psychiatry5714451450199410.1136/jnnp.57.12.14457798970 Levi-MontalciniRThe nerve growth factor 35 years laterScience23711541162198710.1126/science.33069163306916 ThoenenHBandtlowCHeumannRThe physiological function of nerve growth factor in the central nervous system: Comparison with the peripheryRev Physiol Biochem Pharmacol109145178198710.1007/BFb00310263317757 BardeYANeurotrophins: A family of proteins supporting the survival of neuronsProg Clin Biol Res390455619947724649 SofroniewMVHoweCLMobleyWCNerve growth factor signaling, neuroprotection, and neural repairAnnu Rev Neurosci2412171281200110.1146/annurev.neuro.24.1.121711520933 KorschingSAuburgerGHeumannRScottJThoenenHLevels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervationEmbo J41389139319852411537 ConnorBDragunowMThe role of neuronal growth factors in neurodegenerative disorders of the human brainBrain Res Brain Res Rev27139199810.1016/S0165-0173(98)00004-69639663 Levi-MontalciniAAngelettiPUNerve growth factorPhysiol Rev4853456919684875350 ChaoMVHempsteadBLp75 and Trk: A two-receptor systemTrends Neurosci18321326199510.1016/0166-2236(95)93922-K7571013 ZhangZYangYGongAWangCLiangYChenYLocalization of NGF and TrkA at mitotic apparatus in human astrocytoma cell line u251Biochem Biophys Res Commun3376874200510.1016/j.bbrc.2005.08.26516181609 FriedmanWJIbáñezCFHallböökFPerssonHCainLDDreyfusCFBlackIBDifferential actions of neurotrophins in the locus corelueus and basal forebrainExp Neurol1197278199310.1006/exnr.1993.10078432352 MamounasLABlueMESiuciakJAAltarCABrain-derived neurotrophic factor promotes the survival and sprouting of serotonergic axons in rat brainJ Neurosci157929793919958613731 SiuciakJAAltarCAWiegandSJLindsayRMAntinociceptive effect of brain-derived neurotrophic factor and neurotrophin-3Brain Res633326330199410.1016/0006-8993(94)91556-37511037 Sklair-TavronLNestlerEJOpposing effects of morphine and the neurotrophins, NT-3, NT-4, and BDNF, on locus coeruleus neurons in vitroBrain Res702117125199510.1016/0006-8993(95)01029-88846066 JuricDMMiklicSCarman-KrzanMMonoaminergic neuronal activity up-regulates BDNF synthesis in cultured neonatal rat astrocytesBrain Res11085462200610.1016/j.brainres.2006.06.00816828062 BalohRHEnomotoHJohnsonEMJrMilbrandtJThe GDNF family ligands and receptors-implications for neural developmentCurr Opin Neurobiol10103110200010.1016/S0959-4388(99)00048-310679429 YangZFHoDWLauCKTamKHLamCTYuWCPoonRTFanSTSignificance of the serum brain-derived neurotrophic factor and platelets in hepatocellular carcinomaOncol Rep1612371243200617089044 Tapia-ArancibiaLRageFGivaloisLArancibiaSPhysiology of BDNF: Focus on hypothalamic functionFront Neuroendocrinol2577107200410.1016/j.yfrne.2004.04.00115571756 MatsumotoKYamamotoKKarasawaYHinoNNakamuraATakahashiMArakiHOkuyamaSChoshiTSuginoEPossible involvement of induction of brain-derived neurotrophic factor in the neuroprotective effect of a 5-phenylpyrimidine derivativeBiochem Pharmacol6610191023200310.1016/S0006-2952(03)00462-312963488 RennéCWillenbrockKKüppersRHansmannMLBräuningerAAutocrine and paracrine-activated receptor tyrosine kinases in classic hodgkin lymphomaBlood10540514059200510.1182/blood-2004-10-400815677564 PearseRNSwendemanSLLiYRafiiDHempsteadBLA neurotrophin axis in myeloma: TrkB and BDNF promote tumor-cell survivalBlood10544294436200510.1182/blood-2004-08-309615657181 DoumaSVan LaarTZevenhovenJMeuwissenRVan GarderenEPeeperDSSuppression of anoikis and induction of metastasis by the neurotrophic receptor TrkBNature43010341039200410.1038/nature0276515329723 GrotzerMAJanssAJFungKBiegelJASuttonLNRorkeLBZhaoHCnaanAPhillipsPCLeeVMTrojanowskiJQTrkC expression predicts good clinical outcome in primitive neuroectodermal brain tumorsJ Clin Oncol1810271035200010.1200/JCO.2000.18.5.102710694553 SegalRAGoumnerovaLCKwonYKStilesCDPomeroySLExpression of the neurotrophin receptor TrkC is linked to a favorable outcome in medulloblastomaProc Natl Acad Sci USA911286712871199410.1073/pnas.91.26.128677809137 ChiappaSAChinLSZurawelRHRaffelCNeurotrophins and Trk receptors in primitive neuroectodermal tumor cell linesNeurosurgery4511481155199910.1097/00006123-199911000-0002610549931 Colucci-D'AmatoGLD'AlessioACalifanoDCaliGRizzoCNitschLSantelliGde FranciscisVAbrogation of nerve growth factor-induced terminal differentiation by ret oncogene involves perturbation of nuclear translocation of ERKJ Biol Chem2751930619314200010.1074/jbc.275.25.1930610858459 Devouassoux-ShisheboranMMauduitCTaboneEDrozJPBenahmedMGrowth regulatory factors and signalling proteins in testicular germ cell tumoursAPMIS111212224200310.1034/j.1600-0463.2003.11101251.x12752264 EggertASievertsHIkegakiNBrodeurGMP75 mediated apoptosis in neuroblastoma cells is inhibited by expression of TrkAMed Pediatr Oncol35573576200010.1002/1096-911X(20001201)35:6<573::AID-MPO17>3.0.CO;2-A11107120 GoustinASLeofEBShipleyGDMosesHLGrowth factors and cancerCancer Res461015102919863002607 NishioSMoriokaTHamadaYFukuiMNakagawaraAImmunohistochemical expression of tyrosine kinase (Trk) receptor proteins in mature neuronal cell tumors of the central nervous systemClin Neuropathol1712313019989625303 ZhuZFriessHdiMolaFFZimmermannAGraberHUKorcMBüchlerMWNerve growth factor expression correlates with perineural invasion and pain in human pancreatic cancerJ Clin Oncol1724192428199910.1200/JCO.1999.17.8.241910561305 ZhuZWFriessHWangLDi MolaFFZimmermannABüchlerMWDown-regulation of nerve growth factor in poorly differentiated and advanced human esophageal cancerAnticancer Res20125132200010769644 DolléLElYazidi-BelkouraIAdriaenssensENurcombeVHondermarckHNerve growth factor overexpression and autocrine loop in breast cancer cellsOncogene2255925601200310.1038/sj.onc.120680512944907 BreitSAshmanKWiltingJRösslerJHatziEFotsisTSchweigererLThe N-myc oncogene in human neuroblastoma cells: Down-regulation of an angiogenesis inhibitor identified as activinCancer Res6045964601200010969812 ZhangJZhangJLiAFanYRole of nerve growth factor receptor in neuroblastoma angiogenesisChinese J Contemporary Pediatrics693982004 HuYSunCYWangHFGuoTWeiWNWangYDHeWJWuTTanHWuTCBrain-derived neurotrophic factor promotes growth and migration of multiple myeloma cellsCancer Genet Cytogenet1691220200610.1016/j.cancergencyto.2006.02.01816875931 QiuLZhouCSunYDiWSchefflerEHealeySKouttabNChuWWanYCrosstalk between EGFR and TrkB enhances ovarian cancer cell migration and proliferationInt J Oncol2910031011200616964397

NGF was observed in the cytoplasm and nucleus of normal brain tissue and astrocytomas, but primarily in the nucleus. The expression of NGF in (A) normal brain tissue, (B) grade I astrocytoma, (C) grade II astrocytoma, (D) grade III astrocytoma and (E) grade IV astrocytoma. Magnification, ×400. NGF, nerve growth factor.

BDNF was observed in the cytoplasm of normal brain tissues and astrocytomas. The expression of the BDNF in (A) normal brain tissue (magnification, ×400), (B) grade I astrocytoma (magnification, ×200), (C) grade II astrocytoma magnification, ×200), (D) grade III astrocytoma (magnification, ×400) and (E) grade IV astrocytoma (magnification, ×400). BDNF, brain-derived neurotrophic factor.

Expression of NGF and BDNF in normal brain tissue and astrocytomas of distinct pathological grades.

Grade Cases NGF expression, % BDNF expression, %
Normal tissues 15 7.06±0.95a 5.98±0.49a
I 10 16.56±4.56a 11.24±2.05a
II 26 32.45±10.07a 18.23±4.96a
III 24 40.91±26.40a 21.44±3.35a
IV 10 24.71±8.28a 15.39±2.01a

Statistically significant difference among compared groups (P<0.05). NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor.

Expression of NGF and BDNF from distinct astrocytoma locations.

Region Cases NGF expression, % BDNF expression, %
Temporal lobe 18 42.57±13.57a 24.19±7.12a
Parietal lobe 16 35.62±9.47a 20.09±4.56a
Cerebellum 16 28.67±6.47a 16.17±4.29a
Frontal lobe 20 21.45±7.56a 16.17±4.29a

Statistically significant difference among compared groups (P<0.05). NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor.