MORFOLOGI DAN PERSENTASE NEURON DOPAMINERGIK DARI KULTUR VENTRAL MESENSEFALON FETUS TIKUS PUTIH YANG DIPLATTING DALAM MINIMUM ESSENTIAL MEDIUM + 10% HORSE SERUM MENGGUNAKAN PEWARNAAN IMUNOSITOKIMIA DENGAN PENANDA TIROSIN HIDROKSILASE KONSENTRASI 1:1000

TUTI TRI SEDYA R TYAS, Dr. drh. Tri Wahyu Pangestiningsih, MP.

2016 | Skripsi | S1 KEDOKTERAN HEWAN

Penyakit Parkinson merupakan penyakit neurodegeneratif karena rusaknya neuron dopaminergik di substansia nigra pars compacta (SNpc). Metode pengobatan yang tengah dikembangkan adalah exogenous cell replacement, namun terkendala karena jumlah donor jaringannya terbatas. Penelitian ini bertujuan mengetahui gambaran mikroskopik berupa persentase dan morfologi kultur neuron dopaminergik dari ventral mesensefalon fetus tikus putih umur 14 hari dengan pewarnaan imunositokimia pada media minimum essential medium (MEM) + 10% horse serum (HS) yang diharapkan mendapatkan metode kultur neuron dopaminergik yang optimal dan sederhana. Ventral mesensefalon fetus tikus putih (Rattus norvegicus albinus) umur 14 hari dikoleksi pada tabung konikal berisi media MEM + 10% HS + 10% fetal bovine serum, kemudian didisosiasi secara mekanik dengan mikropipet, disentrifugasi selama lima menit pada kecepatan 200 g sebanyak dua kali, selanjutnya dipindahkan ke kultur primer (flask) dengan media yang sama sebanyak 2 ml lalu diinkubasi selama 60 menit dengan kadar CO2 5% pada suhu 37oC, kemudian ditambah 3 ml media dan diinkubasi kembali selama tiga hari. Kultur diplatting pada plat berlapis kolagen 96 sumuran dengan media MEM + 10% HS selama tiga hari, dilanjutkan dengan pewarnaan imunositokimia metode Labeled Streptavidin-biotin (LSAB) dengan antibodi primer anti-Tirosin Hidroksilase rabbit serum (1:1000,Chemicon Int., Temecula, USA Cat No. AB152). Morfologi neuron dopaminergik diamati menggunakan mikroskop inverted perbesaran 10x20 dan dilakukan penghitungan persentase neuron dopaminergik terhadap seluruh sel yang tumbuh dengan perbesaran 10x10 pada 10 area acak tiap sumuran secara manual oleh tiga orang. Persentase neuron dopaminergik yang tumbuh pada media adalah 10,4 ±14,2%/area. Morfologi neuron pada kultur yang teramati adalah neuron unipolar, neuron bipolar dan neuron multipolar.

Parkinsons disease is a neurodegenerative disease caused by progressive loss of the dopaminergic neuron in the substansia nigra pars compacta (SNpc). Exogenous cell replacement currently being developed as a treatment of Parkinsons disease, but the main problem remains the limited supply of donor tissue. The aim of this research is to find out morphology and percentage the dopaminergic neurons culture of fetal day 14 (Fd14) rat ventral mesencephalic using immunocytochemistry staining in minimum essential medium (MEM) + 10% horse serum (HS) media which is expected to optimize culture methods. Ventral mesencephalon of Fd14 rat (Rattus norvegicus albinus) was collected and cultured in conical tube containing MEM + 10% HS + 10% fetal bovine serum media, then dissosiated mechanically using micropipette. The tissue was centrifuged for five minutes at 200 g twice, then transferred to a primary cuture (flask) containing the same media then incubated for 60 minutes at 37oC with 5% CO2 level, afterwards 3 ml media was added to the culture and incubated for three days. Cultured cells then being platted on collagen-coated 96-wells tissue culture plate with MEM + 10% HS media for three days and then immunocytochemistry staining was done using Labeled Streptavidin-biotin (LSAB) method with primary anti-tirosine hydroxylase antibody rabbit serum(1:1000, Chemicon Int., Temecula, USA Cat No. AB152). The neuron morphology was observed using inverted microscope with 10x20 magnification and the percentage number of dopaminergic neurons compared to entire grown cells was counted with 10x10 magnification on 10 areas/wells randomly by three persons. The result showed that the percentage of dopaminergic neurons succesfully grown in the media is 10,4 ±14,2%/area. The morphology of cultured neurons found were unipolar neuron, bipolar neuron and multipolar neuron.

Kata Kunci : imunositokimia, kultur neuron dopaminergik, tirosin hidroksilase, ventral mesensefalon