Analisis regenerasi Medulla Spinalis ekor kadal (Mabuya multifasciata Kuhl)

SOESILO, Nyoman Puniawati, Promotor Prof.Dr. Soesanto Mangkoewidjojo, M.Sc

2006 | Disertasi | S3 MIPA (Biologi)

Proses regenerasi ekor kadal sangat tergantung pada medulla spinalis dan angiogenesis. Selama regenerasi ekor terjadi regenerasi medulla spinalis, walaupun struktur regenerat medulla spinalis tidak sempurna. Tetapi struktur regenerat medulla spinalis, neuron yang aksonnya mencapai regenerat medulla spinalis dan kemampuan medulla spinalis diproksimal regenerat ekor dalam menstimulasi angiogenesis belum diketahui. Penelitian ini bertujuan untuk mengetahui struktur medulla spinalis pada regenerat ekor kadal, neuron yang aksonnya mencapai regenerat medulla spinalis dan kemampuan medulla spinalis ekor dalam memicu angiogenesis. Diduga struktur regenerat medulla spinalis berbeda dengan medulla spinalis ekor asli, akson yang terdapat pada regenerat medulla spinalis berasal dari neuron intraspinalis dan supraspinalis, medulla spinalis di proksimal regenerat ekor berbagai fase regenerasi mampu menstimulasi angiogenesis Struktur regenerat medulla spinalis dipelajari dengan mikroskop cahaya dan mikroskop elektron. Neuron yang merupakan asal akson yang terdapat dalam regenerat medulla spinalis dideteksi dengan metode axonal retrograde transport, dengan Horseradish peroxidase (HRP)sebagai penanda. Kemampuan medulla spinalis menstimulasi angiogenesis diamati secara in vitro dengan melakukan implantasi medulla spinalis bagian proksimal regenerat ekor semua fase regenerasi pada membran korioalantois (MKA) embrio ayam umur 8 hari. Untuk mengetahui faktor angiogenik yang terlibat dalam proses regenerasi, dilakukan analisis profil protein medulla spinalis tersebut dengan teknik SDS-PAGE. Hasil pengamatan menunjukkan terdapat perbedaan struktur regenerat medulla spinalis dan medulla spinalis ekor asli terutama pada sel ependima dan akson. Pada medulla spinalis ekor asli, sel ependima berbentuk kubus atau kolumnar rendah tanpa ada tonjolan basal sedangkan pada regenerat medulla spinalis, sel ependima berbentuk kolumnar tinggi dilengkapi dengan tonjolan basal meluas ke arah lamina basal. Tonjolan basal sel ependima yang berdekatan membentuk bangunan berupa saluran, dilalui oleh akson yang mengalami regenerasi. Akson tersebut sebagian besar tidak bermyelin dan berasal dari neuron intraspinalis. Baik pada sel ependima medulla spinalis ekor asli maupun sel ependima regenerat medulla spinalis, organel yang terlihat jelas dalam jumlah banyak adalah mitokondria, kompleks Golgi dan ribosoma bebas, sedangkan retikulum endoplasmik granular terlihat sedikit di dekat inti. Medulla spinalis proksimal regenerat ekor fase penyembuhan luka dan diferensiasi paling aktif menstimulasi angiogenesis (skor 2+), medulla spinalis proksimal regenerat ekor fase morfogenesis, pertumbuhan dan matang regenerasi kurang aktif (skor 1+), sedangkan medulla spinalis ekor normal tidak menstimulasi angiogenesis (skor 0). Profil protein medulla spinalis menunjukkan pita protein berat molekul 39 kD dan 43 kD terlihat lebih tebal (memiliki konsentrasi protein yang lebih tinggi) pada medulla spinalis proksimal regenerat ekor fase penyembuhan luka dan fase diferensiasi dibandingkan dengan konsentrasi protein yang sama pada medulla spinalis regenerat ekor fase regenerasi yang lain. Dari hasil penelitian ini dapat disimpulkan bahwa struktur regenerat medulla spinalis berbeda dengan struktur medulla spinalis ekor asli; serabutserabut syaraf yang terdapat pada regenerat medulla spinalis berasal dari neuron intraspinalis; medulla spinalis proksimal regenerat ekor fase penyembuhan luka dan fase diferensiasi paling aktif menstimulasi angiogenesis. Konsentrasi protein berat molekul 39 kD dan 43 kD pada nilai ambang tertentu mempunyai kemampuan sangat aktif dalam menstimulasi angiogenesis. Protein tersebut diduga sebagai salah satu faktor angiogenik dalam proses regenerasi ekor kadal.

Tail regeneration in the lizard is dependent upon the presence of spinal cord and angiogenesis. During regeneration, lizard also regenerate the tail spinal cord, although the regenerated spinal cord is imperfect. However, the structure of the regenerated tail spinal cord, the origin of the axons in the regenerated spinal cord and the ability of the spinal cord to stimulate angiogenesis were not known. The main goals of this study were to describe morphologic structure of the regenerated spinal cord of lizard’s tail, to identify the origin of the axons in the regenerated spinal cord, and to observe the ability of lizard’s tail spinal cord to promote the angiogenesis. It was suggested that the regenerated and the original spinal cord have difference structure, the axons in the regenerated spinal cord have supraspinal and intraspinal origin and the spinal cord was able to stimulate the angiogenesis. The structure of the regenerated spinal cord was examined by light and electron microscopy. The neurons from which axons originated in the regenerated spinal cord were detected by axonal retrograde transport techniques. Angiogenesis response was observed in vitro by implanting the spinal cord tissue of proximal to tail regenerate of all regeneration phases, on the chorioallantoic membrane (CAM) of eight day chick embryos. The SDS-PAGE techniques were used to analyze protein profile of the spinal cord. The results showed that the ependymal cells of the original spinal cord appeared to be cuboidal to low columnar, without the basal processes. The ependymal cells of the regenerated spinal cord were high columnar with the ependymal processes extending from the basal portion of the cells toward the piamater. Numerous mitochondria, Golgi complex and free ribosome were present in the cytoplasm of ependymal cells of either the original or regenerated spinal cord, and limited rough endoplasmic reticulum were shown close to the nucleus. The majority of the regenerated axons were nonmyelinated and were located between the basal processes of the adjacent ependymal cells. This study demonstrated that only a small number of supraspinal neurons extend axons into the regenerated spinal cord, however, the majority of the axons in the regenerated spinal cord were local spinal (intraspinal) origin. As the spinal cords were implanted on the CAM, new blood vessels were converging toward the implant. The most active of angiogenesis occurred in the CAM implanted with spinal cords proximal to the regenerate of wound healing and differentiation phase (Score 2+), and these spinal cords had similar protein profiles. However a protein profile of 39 kD and 43 kD of spinal cord proximal to the regenerat of wound healing and differentiation phases, resulted a thicker band than that were found in other phases of regeneration. It was concluded that the regenerated and the original spinal cord have difference structure, the axons in the regenerated spinal cord have intraspinal origin; the spinal cord proximal to the regenerate of wound healing and differentiation phases were able to stimulate angiogenesis and they have higher concentration of protein (39 kD and 43 kD) than the same protein of spinal cord proximal to the regenerat of other regeneration phases. This study suggested that the protein of 39 kD and 43 kD in the lizard spinal cord represented one of angiogenesis factor which plays role in the regeneration processes.

Kata Kunci : Kadal,Regenerasi Ekor,Medulla Spinalis, lizard, spinal cord, regeneration, retrograde transport, angiogenesis