ADLN Perpustakaan Universitas Airlangga

ADLN Perpustakaan Universitas Airlangga

PENINGKATAN L-TYPE CALCIUM CHANNEL, KALSIUM INTRASELULER, PAR-1 dan HSP-27 pada UTERUS KELINCI NEW ZEALAND YANG DILAKUKAN PENJAHITAN MODIFIKASI SURABAYA (KAJIAN BIOMOLEKULER PADA PENANGANAN PERDARAHAN PASCA PERSALINAN KARENA ATONIA UTERI) SULISTYONO, AGUS

Promotor: Prof. Dr. Kuntoro, dr, MPH INTRACELLULAR CALCIUM KKA KK Dis K 53/12 Sul p Copyright© 2012 by Airlangga University Library Surabaya

RINGKASAN Setiap tahun terdapat 529.000 kematian maternal dan 99% terjadi di negara berkembang, penyebab utamanya adalah perdarahan pasca persalinan (PPP) yaitu sebanyak 25%. Berdasarkan Survei Demografi dan Kesehatan Indonesia pada tahun 1994 terdapat kematian maternal sebanyak 390 per 100.000 kelahiran hidup, tahun 2001 menurun menjadi 307 dan tahun 2007 sebanyak 248. Target angka kematian maternal Millenium Development Goal tahun 2015 menjadi 100 kematian maternal per 100.000 kelahiran hidup. Terdapat 4 penyebab utama PPP, atonia uteri merupakan penyebab terbanyak yaitu 70%, sisanya karena laserasi jalan lahir, kelainan penempelan plasenta dan kelainan darah. Penanganan PPP yang paling penting adalah menghentikan perdarahan sambil memperbaiki keadaan umum ibu, menentukan etiologinya dan memberikan terapi yang sesuai. Kegagalan menentukan etiologi dan penanganan yang tepat menyebabkan perdarahan tidak berhenti dan menimbulkan penyulit yang sulit diatasi. Mekanisme penghentian perdarahan pada PPP berbeda dengan organ lain yang tergantung vasospasme pembuluh darah dan pembekuan darah. Pada PPP penghentian perdarahan terutama karena kontraksi dan retraksi miometrium sehingga menyempitkan dan membuntu lumen pembuluh darah. Bila kontraksi dan retraksi tidak berfungsi dengan baik, perdarahan tidak akan berhenti walaupun faktor pembeku darah normal, sebaliknya walaupun faal pembeku darah tidak baik PPP dapat berhenti asalkan kontraksi dan retraksi uterus berfungsi normal. Kontraksi miometrium dipengaruhi oleh peningkatan kalsium intraseluler yang terutama berasal dari influks melalui L-type calcium channel. Kalsium intraseluler berikatan dengan reseptornya, yaitu kalmodulin membentuk kompleks kalsium kalmodulin yang dapat mengaktivasi Myosin Light Chain Kinase (MLC Kinase) menjadikan MLC aktif sehingga bisa berikatan dengan aktin-F yang merupakan bentuk aktif aktin. Aktin-F berasal dari remodeling aktin-G (tidak aktif) dengan bantuan Heat Shock Protein 27 (HSP-27) yang telah terfosforilasi. Fosforilasi HSP-27 melalui jalur Mitogen Activated Protein Kinase (MAPK) yang terjadi karena adanya enzim phospholipase-C (PLC). PLC meningkat karena adanya rangsangan mekanis atau agonis kontraksi pada protein G di membran miometrium. Penjahitan kompresi uterus modifikasi Surabaya dengan menggunakan 3 jahitan vertikal uterus yang merupakan modifikasi jahitan kompresi metode B-Lynch, adalah salah satu metode penanganan operatif konservasi uterus untuk menghentikan PPP karena atonia uteri yang sederhana, cepat, mudah dan efektif. Penjahitan kompresi uterus bekerja seperti kompresi bimanual, menyebabkan uterus mengecil dan cavum uteri tertutup sehingga perdarahan segera berhenti. Berhentinya perdarahan yang semula profus, dapat memberi kesempatan terbentuknya trombin. Trombin berikatan dengan reseptornya, yaitu Protease Activating Receptor-1 (PAR-1) dan mengaktifkan protein G yang selanjutnya melalui jalur PLC menimbulkan kontraksi miometrium. Di samping itu jahitan kompresi sendiri merupakan Desertasi

PENINGKATAN L-TYPE CALCIUM CHANNEL....

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rangsangan mekano-transduksi yang dapat menimbulkan depolarisasi membran sel sehingga L-type calcium channel membuka dan juga mengaktifkan protein G. Untuk mengkonfirmasi terjadi kontraksi miometrium setelah dilakukan penjahitan kompresi metode Surabaya, dilakukan penelitian eksperimental laboratorium secara cross sectional, single blinded dengan interval waktu ½, 1, 2 dan 24 jam pada model uterus kelinci bunting aterm yang dilakukan operasi pengeluaran janin, irisan uterus dijahit kembali, selanjutnya pada uterus ipsilateral dilakukan penjahitan kompresi modifikasi Surabaya sedang sisi kontralateralnya sebagai kontrol tidak dilakukan. Variabel kontraktilitas miometrium yang diteliti adalah ekspresi L-type calcium channel, PAR-1 dan HSP-27 yang diperiksa secara imunohistokimia serta densitas kalsium intraseluler berdasar intensitasnya di sitoplasma yang diperiksa dengan cara fluoro 3 double staining yang diamati dengan mikroskop konfokal. Pada penelitian ini didapatkan peningkatan secara bermakna ekspresi L-type calcium channel pada semua interval waktu, yaitu pada pengamatan ½ jam dari kontrol dan perlakuan didapat hasil 2,78±1,39 vs 12,89±5,30 (p=0,001); 1 jam 3,93±0,49 vs 14,87±1,05 (p<0,0001); 2 jam 1,48±0,46 vs 10,01±3,21 (p=0,011) dan 24 jam 2,95±0,18 vs 10,01±3,21 (p<0,0001) sel/ 100 sel perlapang pandang. Didapatkan juga peningkatan yang bermakna pada densitas kalsium intraseluler pada ke 4 interval waktu, yaitu 1722,52±769,30 vs 2375,26±264,53 (p=0,22); 1721,60±426,93 vs 2467,84±209,13 (p=0,001); 1722,78±449,92 vs 2233,24±283,73 (p=0,005) dan 1527,52±167,63 vs 1988,61±293,73 (p=0,002) intensitas/μm2. Ekspresi PAR-1 pada semua interval waktu juga meningkat secara bermakna, yaitu 5,39±1,18 vs 16,36±1,31 (p<0,0001); 12,34±1,81 vs 23,72±2,80 (p<0,0001); 12,71,1,60 vs 23,42±1,38 (p<0,0001) dan 12,55±1,60 vs 22,97±1,35 (p<0,0001) sel perlapang pandang. Pada ekspresi HSP-27 juga didapatkan peningkatan yang bermakna antara kelompok kontrol dibanding perlakuan pada semua interval waktu, 3,82±1,49 vs 13,15±2,99 (p<0,0001); 4,93±2,67 vs 22,45±4,06 (p<0,0001); 5,20±1,71 vs 21,09±4,06 (p<0,0001) dan 5,41±1,73 vs 21,09±1,92 (p<0,0001) sel perlapang pandang. Dari pengamatan masingmasing variabel pada interval waktu pada kelompok kontrol didapatkan pola nilai variabel yang meningkat dari ½ jam ke 1 jam, kemudian plateau atau sedikit meningkat / menurun pada jam ke 2 dan plateau lagi atau menurun pada jam ke 24. Gambaran / pola serupa didapatkan juga pada kelompok perlakuan. Dari hasil penelitian dapat disimpulkan bahwa penjahitan kompresi uterus modifikasi Surabaya meningkatkan ekspresi L-type calcium channel, PAR-1, HSP-27 dan densitas kalsium intraseluler secara bermakna dengan tanpa mengubah pola perubahan variabel terperiksa. Peningkatan semua variabel prokontraktil memicu timbulnya kontraksi miometrium yang berdampak penghentian perdarahan pada PPP.

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SULISTYONO, AGUS


SUMMARY THE INCREASED EXPRESSION OF L-TYPE CALCIUM CHANNEL, PAR-1 AND HSP-27 AND DENSITY OF INTRACELLULAR CALCIUM ON ANIMAL MODEL OF NEW ZEALAND RABBIT MYOMETRIUM THAT USING CONSERVATIVE SURGICAL PROCEDURE OF SURABAYA SUTURE COMPRESSION MODIFICATION (BIOMOLECULAR STUDY IN CONSERVATIVE SURGICAL MANAGEMENT OF PPH DUE TO UTERINE ATONY) AGUS SULISTYONO Postpartum hemorrhage (PPH) is the leading cause of maternal mortality and suggested that 25% of 529,000 maternal death per year in the world due to PPH. Ninety nine percent of maternal mortality occur in developing countries. According Indonesia Health and Demography Survey (SDKI) there are 390 maternal mortalities per 100,000 live births in 1994 (maternal mortality ratio=MMR) and decreasing to 307 maternal mortalities in 2001 and 248 in 2007. Target of the Millenium Development Goals in the year of 2015 is 100 of MMR. There are 4 T’s etiologies of PPH, Tone, as the leading cause, uterine atony, failure of uterine to contract and retract following delivery of the baby, accounts for about 70%, Trauma, traumatic injury to the genital tract (about 20%), Tissue, retained placenta (about 10%) and Thrombin, coagulation disorders (very rare). The treatment of PPH patient has 2 major components, the first are give resuscitation and stop bleeding, and the second are identification and management of the underlying cause(s) of the hemorrhage. However the successful management of PPH requires that both components be simultaneously and systematically addressed. Unlike another organ of the body, the uterus is very unique organ, because the most important for achieving hemostasis at placental site bleeding are contraction and retraction of myometrium to compress the formidable number of relative large vessels and obliterate their lumen. PPH can be fatal if it occur in hypotonic uterus, although the maternal blood coagulation is normal. Conversely, if the myometrium at placental implantation site contract and retract vigorously, profuse PPH is unlikely even the coagulation mechanism is impaired. The basic nature of uterine contraction is critically dependent on the increasing of intracellular calcium due to calcium influx through L-type calcium channel. Intracellular calcium bind to its receptor, calmodullin and the calcium calmodullin complex will activate Myosin Light Chain Kinase (MLCK). Binding of calmodulin to MLCK results in an activation of this kinase and phosphorolation of MLC and triggers myometrial contraction. Mechanical stimuli or the binding of contractile agonist to G-protein leads to activation Gq subunit and activates phospholipase C (PLC). This enzyme catalyzes PIP2 to IP3 and DAG. IP3 induces the release of Calcium from Sarcoplasmic Reticulum, while DAG activates protein kinase C (PKC) and MAPK pathways and phosphorilates HSP-27. HSP-27 induces actin remodeling, from Globular actin (G-actin) to Fibrillar actin (F-actin). Interaction of phosphorilated MLC with F-actin induce myometrial contraction and retraction. The Surabaya compression suturing modification, a modified B-Lynch brace suturing technique is a new conservative surgical technique preserving uterus with 3 vertical compression suture which simpler, quicker and effective. The Surabaya methods, like another B-Lynch modification, compress uterine atony to make uterus smaller and obliterate the uterine arteries in myometrium and to stop bleeding from implantation placental site. Uterine compression sutures using Surabaya modification succeed to stop Desertasi


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bleeding in many cases of PPH due to atony. The aims of this study is to confirm increasing L-type calcium channel, intracellular calcium, PAR-1 and HSP-27 in Surabaya compression suturing as a mechanotransducer biomolecular pathways to induce myometrial contraction. The study was done from March to August 2011, true experimental, single blinded with time interval were ½, 1, 2 and 24 hours, using term uterine pregnant New Zealand rabbits. Its consists 11 uterine samples of Surabaya methods (cases) and 11 samples without compression suturing technique as controls. The result of this study is significantly increase of all variables observed in cases compare to control groups at 4 time intervals, ½, 1, 2 and 24 hours. At ½ hour observation, the expression of L-type calcium channel are 2,78±1,39 vs 12,89±5,30 (p=0,001); 1 hour 3,93±0,49 vs 14,87±1,05 (p<0,0001); 2 hour 1,48±0,46 vs 10,01±3,21 (p=0,011) dan 24 hour are 2,95±0,18 vs 10,01±3,21 (p<0,0001) cell/100 cell observational field. The densities of intracellular calcium at ½ hour are 1722,52±769,30 vs 2375,26±264,53(p=0,22); at 1 hour observation are 1721,60±426,93 vs 2467,84±209,13 (p=0,001); at 2 hours are 1722,78±449,92 vs 2233,24±283,73 (p=0,005) and at 24 hours are 1527,52±167,63 vs 1988,61±293,73 (p=0,002) intensities/μm2. Expression of PAR-1 are 5,39±1,18 vs 16,36±1,31 (p<0,0001); 12,34±1,81 vs 23,72±2,80 (p<0,0001); 12,71,1,60 vs 23,42±1,38 (p<0,0001) and 12,55±1,60 vs 22,97±1,35 (p<0,0001) cells/observational field. HSP-27 expression are 3,82±1,49 vs 13,15±2,99 (p<0,0001); 4,93±2,67 vs 22,45±4,06 (p<0,0001); 5,20±1,71 vs 21,09±4,06 (p<0,0001) dan 5,41±1,73 vs 21,09±1,92 (p<0,0001) cells/observational field. Conclusion of this study is uterine compression suture (Surabaya modification suture) increasing all variables, L-type calcium channel, PAR-1, HSP-27 expression and calcium intracellular density compare to control. Mechanotransduction of uterine compression trigger uterine myocyte contractility pathway to induce myometrial contraction.

ABSTRACT THE INCREASED EXPRESSION OF L-TYPE CALCIUM CHANNEL, PAR-1 AND HSP-27 AND DENSITY OF INTRACELLULAR CALCIUM ON ANIMAL MODEL OF NEW ZEALAND RABBIT MYOMETRIUM THAT USING CONSERVATIVE SURGICAL PROCEDURE OF SURABAYA SUTURE COMPRESSION MODIFICATION (BIOMOLECULAR STUDY IN CONSERVATIVE SURGICAL MANAGEMENT OF PPH DUE TO UTERINE ATONY) AGUS SULISTYONO Objective : Uterine atony as the common cause of PPH which causing high maternal mortality can indeed be largely rescued by compression with conservative surgical procedure by performing Surabaya suture compression modification (SSCM). To unveil the biomolecular phenomena after Surabaya compression suture leading myometrium contraction, the purpose of this study is to analyze the increased expression Desertasi


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of L-type calcium channel, PAR-1 and HSP-27 and density of intracellular calcium on animal model of New Zealand rabbit myometrium after procedure performance Material and methods: True experimental single blinded study was done in Airlangga University Veterinary Faculty and Brawijaya University Physiology Department from March – August 2011. Twenty two samples were obtained over this study which applied on aterm pregnant rabbit uterus as animal model. Animal model consisted of rabbit uterus in which practiced on by SSCM in 11 samples after terminating pregnancy and removing pup and placenta as well, and the remainder of another 11 samples without SSCM as the control. The assessment of L-type calcium channel, PAR-1 and HSP-27 expression and density of intracellular calcium on myometrium sample was done respectively by immunohistochemistry and fluoro-2 double staining method at ½,1,2, and 24 hours intervals Result: At all 4 time intervals the increased expression of L-type calcium channel was observed significantly in SSCM compared to control (p=0.001; p< 0.0001; p=0.011 and p<0.0001), also for intracellular calcium density (p=0.022; p=0.001;p=0.005 and p=0.002). The study showed PAR-1 and HSP-27 expression increasing sharply that statistically analysis indicated both all of p similarly < 0.0001 against control. Conclusion: SSCM suggested strongly increasing some component (all variable) that absolutely required to myometrium contraction

Keywords: PPH, Surabaya suture compression modification, L-type calcium channel, PAR-1, HSP-27 and intracellular calcium.

Desertasi


SULISTYONO, AGUS

ADLN Perpustakaan Universitas Airlangga

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