An improved primary culture method for hippocampal neurons in fetal rats and MAP2 identification

Yi-Ran Shao,Yong Chang DIWU*,Tao Yue

1First Clinical Medical College of Shaanxi University of Chinese Medicine,Xianyang 712046,China

Abstract Objective:To establish a simple,effective and high-purity primary culture method for fetal rat hippocampal neurons.Methods:Wistar rats of gestational age 18 days were taken and the brain tissue was separated under the microscope.Single neuronal cells were obtained by digestion with Brain Dissociation Kit,and then were seeded in cell plates to observe the basic morphologic structure after 24h,3d,and 5d.Immunofluorescence of microtubule associated protein 2 was applied to assess cell purity of the culture.Results:The hippocampal neurons obtained in this culture method are in good condition and grow vigorously.On the 7th day after culture,the purity of neurons was up to 99.62%.Conclusion:The method is simple and effective for obtaining the high-purity and stable neurons.

Key words:Hippocampal neurons,Fetal rats,Primary culture

Introduction

Primary culture of rat hippocampal neurons in vitro has been widely used to reveal cellular mechanisms in neurobiology for 20 years.This technique is used primarily to reveal specific neuronal-related mechanisms of action that are difficult to analyze in the brain.Researchers can analyze cell structures,cell functions and individual protein localization using a variety of biochemical techniques by isolating and culturing individual neurons.Analyzing molecular mechanism of hippocampal neurons reveals advanced functions of the brain,such as memory or learning.Therefore,establishing a good neuronal in vitro culture model plays a crucial role in the study of neurological diseases.

In this study,improved fetal rat hippocampal neuron in vitro culture technology was used in order to provide a good model of neuronal cell culture in vitro for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.The research results are reported below.

Materials and methods

Experimental animals and major reagents

Experimental AnimalWistar rats,female,gestational age 18 days(by observing the vaginal suppository,calculated by calculating the method of pregnancy in rats) [1],were purchased from Xi'an Jiaotong University,School of Medicine Laboratory Animal Center.All animals were bred in an independent ventilation system (IVC)animal room at a SPF laboratory with an animalroom temperature of 20-25℃and a 12-h light-dark cycle of 12 hours with sufficient feeding and drinking.The feeding and experimental procedures are in accordance with the relevant regulations of Experimental Animal Welfare and Ethics Committee of Shan’xi University of Traditional Chinese Medicine.The experiment was approved by Animal Ethics Committee of Shan’xi University of Chinese Medicine.

Main experimental instruments and materialsClean bench(Tianjin Tesis Instrument Co.,Ltd.);carbon dioxide incubator(HF151VV,Hong Kong Likang Biomedical Technology Co.,Ltd.);Inverted biological microscope(Ningbo Sunny Instrument Co.,Ltd.);Upright automatic fluorescence microscope(Zeiss,Germany);Cell filter screen(200 mesh),cell culture plate(Corning,USA).

Themain reagentD-polylysine(Sigma,USA);D-hank's solution(Hyclone USA);Brain Dissociation Kit digestion solution(Miltenyi Germany);DMEM/F12 medium);Fetal bovine serum(FBS)(Beijing Allgold);Neurobasal(Gibco USA);B27 supplement(Gibco USA);L-glutamine Solution(Beijing Kang century company);microvascular protein-2 antibody(MAP-2),secondary antibody kit(British Abcam company)and so on.

Methods

Pre-treatment of PlatePlates were pre-treated with 0.25% poly-lysine and were placed in CO2 incubator overnight at 37℃,washed with D-hank's solution the next day before use.The 24-well special cell slides were placed in a 24-well plate and then were coated with 200 μl of PDL per well.

Primary Culture of Rat Hippocampal NeuronsThe pregnant rats were executed.Then Open the abdominal cavity along the midline of the abdomen.Open the uterus and take out the fetal rats,which locate in the posterior part of the rat's body cavity.The fetal membrane was stripped and the fetal rats were placed in a petri dish containing pre-cooled d-hank's liquid on the ice.Cut along the fetal rat skull with an eye cut and then curved forceps extrusion of the subcostal cerebral cortex.Turn it under a microscope and remove the pia mater,blood vessels,olfactory bulb,and basal tissue with small forceps to obtain both hemispheres and transfer it into a new Petri dish containing prechilled D-hank's solution on an ice pack.Tissue was then cut into 1 mm3 size with ophthalmic scissors and collected in a 15 ml centrifuge tube.After gently beating for 20 times,5 ml of Brain Dissociation Kit digestion solution prewarmed at 37℃was added and digested in a 37℃water bath for 15 min.Aspirate the digestion solution,add DMEM/F12 stop solution containing FBS(Fetal Bovine Serum)to stop the digestion,repeat 4 times,and repeatedly blow the remaining tissue blocks to disappear.Then the cell suspension was filtered through a 60 μm screen to remove larger cell clumps and centrifuged at 200 g/min for 15 min.The supernatant was discarded and centrifuged once in D-hank's solution to completely terminate the digestion of the Brain Dissociation Kit.Discard the supernatant,add appropriate amount of DMEM/F12 medium carefully re-suspended.Twenty microliters of the mixed cell suspension was dropped on a hemocytometer and counted under an inverted microscope.The cell density was adjusted to(1～10)× 105cells/ml in DMEM/F12 medium and inoculated into polylysine coated over the plate,add the same amount of the same medium,placed in CO2 incubator culture.After 8h,the whole medium was replaced with serum-free Neurobasal medium supplemented with 2%B27 supplement and kept in CO2 incubator.The culture medium was half replaced every 3 days.

Immunofluorescence staining of MAP2Hippocampal neurons cultured for 7 days in vitro were washed twice with PBS at 37℃and fixed with 4% paraformaldehyde at 4℃for 45 minutes and washed with PBS for 3 times for 5 minutes.Adding 0.2%Triton X-100 solution 3 mL permeable cells at room temperature for 10 min,washed with PBS 3 times×5 min.10%goat serum was used to block antigen at room temperature for 30min,washed with PBS 3 times×5min. Dropping MAP2 primary antibody(concentration 1:200),placed in a humid chamber at 4℃overnight.Primary antibody was recovered the next day.After washed with PBS 3 times×5min,the secondary antibody was added and incubated at room temperature for 2h.Then the secondary antibody was recovered and washed with PBS 3 times×5min.At last,4',6-diamidino-2-phenylindole(DAPI)was used to stain nuclei.Use antifluorescence quench sealed tablets before taking pictures.

Results

Morphological observation of rat primary hippocampal neurons

Under a microscope:24h after inoculation,the cells are fully adhered to the wall in a round or oval shape,with 1-3 neurites(Figure 1A).After cultured 3 days,the cells are in fusiform or polygonal shape,with double pole projections.Some cell nucleoli are also clearly visible(Figure 1B). After 5 days culture,it can be seen that the volume of the neurons are further enlarged,the cell bodies become full and the stereoscopic effect is strong with obvious halo around the cells.The shape of neurons becomes various and further form a neural network(Figure 1C).

Figure 1 Morphological observation of rat primary hippocampal neurons after 24h(A),3 days(B)and 5 days(C)culture

Identification and calculation of MAP2 immunofluorescence staining in hippocampus neurons in rats

Primary hippocampal neurons were obtained and labeled with neuronal MAP2 and DAPI immunofluorescence staining.MAP2 was specifically expressed in hippocampal neurons. Under the fluorescence microscope,MAP2 positive cells showed bright green fluorescence,whereas DAPI-positive cells showed blue fluorescence(Figure 2).

Figure 2 Specific identification of the hippocampal neurons

Randomly take 4 fields and calculate the percentage of positive cells in the total cells,which present the purity of primary hippocampal neurons.Our results show that the present of primary hippocampal neurons in the total cells is high,up to 99.62%.

Discussion and conclusion

Primary culture of hippocampal neurons is one of the important techniques to study the structure and function of nervous system.Successful hippocampal neurons cultured by primary culture can be used to provide specific antibodies for target protein,which provide investigators with a mechanism for examining the effect of a single protein on neuronal function.However,although the technology has been more than 20 years old,it is not easy to stably obtain the hippocampal neurons with high purity and good activity for the complicated operation technique.

In this experiment,we modified the previous experimental methods to optimize the growth of healthy neurons while minimizing the contamination of other cells(ie,astrocytes)[2-6].There were four improvements in our research:(1)the tissues were from the embryonic brain tissue of the 18th fetal rats.Compared with the mature brain tissue,the embryonic tissue is easy to detach and contains fewer glial cells,and the neural network is not very rich due to its low degree of differentiation.It is also not easy to destroy the synaptic connections.Therefore,it is easier to get a single pure cell and easy to grow in vitro culture.(2)using neuron-specific digestive enzymes instead of the traditional method of trypsin digestion.Due to the high concentration and unfixed time of trypsin digestion,it is easy to damage the hippocampal neurons.Therefore,in our study,trypsin digestion solution was replaced by digestion solution of Brain Dissociation Kit of German Miltenyi Company.After the follow-up observation,the number of neurons in the hippocampus was increased,the protuberances were longer,the network was dense and the survival rate was high with good growth state.(3)Improvement of cell culture solution and culture solution:In this experiment,DMEM/F12 containing 10%FBS was used as the cell culture solution,and no cytarabine was added to the culture solution.Serum-free Neurobasal medium supplemented with 2%B27 was used as cell culture medium.The main reasons are as follows:①FBS provides neurons special nutrition and it is also conducive to rapid adherent neurons.Cultured with FBS for 8h at first,the neurons adherent firmly,and then by replacing the medium,the glial cells and other impurities cells were removed;②Although cytarabine can reduce the glial cell pollution,but it has certain cytotoxicity on neurons,affecting the vitality of neurons,so this experiment did not use cytarabine;③as a neuron serum-free culture ideal additive,B27 contains the main components(glutamine,insulin,transferrin,etc.)which are the necessary nutrients for cell growth[7-8].Neuralbasal medium is a special basic culture for the neuronal growth which also inhibit the growth of glial cells.It can also be used with B27 to promote the growth of neurons and improve the purity and activity of neurons.(4)Using MAP2 as a neuron-specific marker: MAP2(microtubule-associated protein 2),a cytoskeletal protein that is neuron specific and can be used as an ideal neuron marker[9].The purpose of this study is to develop neurons in hippocampus.Compared with NSE,Anti-Tubulin beta3 and other neuronal markers,the labeling properties and distribution characteristics of MAP2 have been well studied,more stable and more specific.

In this study,using a relatively simple and efficient method,we cultured the primary hippocampal neurons.After 7 days culture,the purity of hippocampal neurons were almost 100%by immunofluorescence cytochemical staining.