Maklumat

Bagaimana cara mengelakkan Bahagian Parafin tidak runtuh?


Saya telah berusaha membuat 5 bahagian saraf tunjang tikus yang cedera dan mengotorkan tisu dengan Luxol Fast Blue dan H&E. Pada mulanya saya mengeluarkan tulang dari sekitar sumsum tulang belakang, tetapi tisu itu runtuh setelah saya mendapatkannya di slaid. Saya rasa kerana ia telah rosak teruk.

Saya fikir saya mungkin dapat mengikat tisu dengan lebih baik jika saya menjaga tulang, jadi saya mengambil tulang belakang dan menyahkalsifikasi dengan 500mM EDTA selama 1 minggu dan mencuba lagi. Tetapi tisu tulang hanya menarik diri dari saraf tunjang.

Ini adalah beberapa gambar saya yang lebih baik. Saya mempunyai banyak orang lain yang masih kurang utuh. Saya melihat tisu runtuh pada slaid semasa proses pengeringan, setelah saya meletakkan bahagian tersebut di permukaan mandi air dan menangkapnya di atas slaid. Saya telah melihat beberapa bahagian tersebar sepanjang slaid. Saya mengesyaki ada kaitan dengan air yang terperangkap di bawah bahagian parafin dan tidak kering dengan betul.

Bagaimana saya boleh memperbaiki prosedur untuk menjaga tisu menjadi 1 bahagian?


Bab 3 - Karsinogenesis paru-paru yang disebabkan oleh uretana

Karsinogenesis yang disebabkan oleh bahan kimia bersama dengan model tikus yang direkayasa secara genetik mewakili pendekatan penting untuk kajian mekanisme kompleks yang melibatkan genotip dan faktor persekitaran dalam perkembangan barah, termasuk barah paru-paru. Induksi tumor paru-paru pada tikus dengan uretana (etil karbamat) dianggap sebagai model berharga Kras-mencegah barah paru-paru. Walau bagaimanapun, strain tikus inbred menunjukkan kerentanan yang berubah-ubah terhadap pembentukan tumor paru-paru, dengan latar belakang C57BL / 6, banyak digunakan untuk mengkaji banyak mutasi transgenik dan nol, sangat tahan terhadap karsinogenesis paru-paru. Di sini dijelaskan protokol kanser paru-paru yang disebabkan oleh uretana yang berkesan dalam induksi tumor paru-paru pada strain C57BL / 6J. Banyak suntikan uretana diperlukan untuk mengatasi ketahanan genetik dan mendorong pembiakan karsinogenesis paru-paru pada tikus latar belakang C57BL / 6J.


Karsinogenesis

J.A. Jones ,. F. Karouia, dalam Toksikologi Komprehensif, 2010

14.10.5.3.1 BRCA1 / 2 Gen

Protein BRCA1 dan BRCA2 multifaktorial mengatur pelbagai fungsi sel, termasuk pembaikan kerosakan DNA, keberadaan, dan peraturan transkrip (Wang et al. 2000). Mutasi pada gen kerentanan kanser payudara dan ovari BRCA1 dan BRCA2 dijumpai pada sebilangan besar keluarga pelbagai kes dengan barah payudara, terutamanya jika mereka juga termasuk satu atau lebih pesakit kes dengan barah ovari (Ford et al. 1995). Pembawa mutasi BRCA1 mempunyai risiko 50-80% untuk menghidap barah payudara pada usia 70 tahun (Easton et al. 1995). Satu salinan BRCA1 atau BRCA2 yang cacat di garis kuman mencukupi untuk kecenderungan barah, tetapi kehilangan alel kedua diperlukan untuk perkembangan barah (Friedman et al. 1994 Miki et al. 1994). Akibat daripada kecacatan ini pada penggabungan semula homolog, tumor yang timbul pada pembawa BRCA cenderung lebih sensitif terhadap sinaran pengionan (Powell dan Kachnic 2003). Yang lain menunjukkan bahawa produk gen BRCA1 / 2 sangat penting untuk mencegah kejadian sekumpulan leukemia dan limfoma (Friedenson 2007). Selaras dengan corak interaksi yang luas ini, mutasi kehilangan fungsi BRCA1 menghasilkan fenotip pleiotrofik, termasuk penundaan pertumbuhan, peningkatan apoptosis, pembaikan kerosakan DNA yang cacat, pendua sentrosom yang tidak normal, titik pemeriksaan kitaran sel G2 / M yang cacat, titik pemeriksaan spindle yang terganggu, dan kerosakan kromosom dan aneuploidi (Brodie dan Deng 2001 Venkitaraman 2002). Fenotip ini tidak serasi, sekurang-kurangnya di permukaan, dengan fungsi penekan tumor yang diberikan kepada BRCA1. Oleh itu, dicadangkan bahawa mutasi pada BRCA1 tidak secara langsung mengakibatkan pembentukan tumor, tetapi sebaliknya menyebabkan ketidakstabilan genetik, menjadikan sel berisiko tinggi terhadap transformasi malignan (Deng 2002 Kinzler dan Vogelstein 1997 Scully dan Livingston 2000). Nampaknya ada konsensus umum bahawa BRCA1 dan BRCA2 didefinisikan sebagai gen penekan tumor, walaupun mekanisme tindakannya masih belum jelas pada masa ini.


Bab 9 Di Situ Analisis Hibridisasi Embrio Anak Ayam di Bahagian Seluruh dan Tisu

Bab ini membentangkan analisis hibridisasi in situ embrio anak ayam di bahagian keseluruhan dan tisu. Pengesanan peraturan temporal dan spasial ekspresi gen pada embrio sangat penting untuk menjelaskan fungsi perkembangan gen dan untuk menjelaskan interaksi sel yang mengatur pola dan pembezaan tisu. Pola ekspresi gen dapat dilihat dengan mengesan produk protein yang dikodkan oleh imunokimia atau RNA messenger menggunakan hibridisasi in situ. Hibridisasi in situ ke RNA melibatkan serangkaian prosedur - yaitu, (1) sintesis probe asid nukleik berlabel yang melengkapi mRNA sasaran, (2) fiksasi dan permeabilisasi tisu, (3) hibridisasi probe ke tisu dan mencuci ke keluarkan probe tidak hibrid, dan (4) pengesanan probe. Banyak jenis probe dan kaedah pelabelan dan visualisasi digunakan untuk hibridisasi in situ kepada embrio. Probe RNA untai tunggal berlabel Hapten paling sering digunakan, kerana ia memungkinkan kepekaan tinggi, resolusi satu sel isyarat, kemampuan untuk memvisualisasikan ekspresi gen pada seluruh embrio, dan pengesanan beberapa RNA dapat dilakukan. Setelah hibridisasi dan pencucian, lokasi probe dikesan dengan protein pengikat hapten yang disambungkan ke enzim. Sejumlah protein hapten dan konjugat enzim protein pengikat hapen tersedia: hapten termasuk digoxigenin, fluorescein, dan dinitrophenol yang dapat dikesan dengan antibodi yang tersedia secara komersial, dan enzim termasuk fosfatase alkali dan peroksidase lobak kuda.


Alamat sekarang: Shionogi Institute for Medical Science, Shionogi & amp Co. Ltd, 12–4 Sagisu 5-Chome, Fukushima-ku, Osaka, 553-0002, Jepun

Gabungan

Jabatan Biokimia dan Biofizik, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, 94143, California, Amerika Syarikat

Gabriele Bergers & Douglas Hanahan

Jabatan Perubatan, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, 94143, California, Amerika Syarikat

Jabatan Anatomi, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, 94143, California, Amerika Syarikat

Institut Penyelidikan Hormon, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, 94143, California, Amerika Syarikat

Gabriele Bergers & Douglas Hanahan

Pusat Kanser Komprehensif UCSF, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, 94143, California, Amerika Syarikat

Zena Werb & amp; Douglas Hanahan

Jabatan Biologi Vaskular, Institut Jantung Harapan, 1124 Columbia Street, Seattle, 98104, Washington, Amerika Syarikat

SUGEN Inc., 230 East Grand Avenue, San Francisco Selatan, 94080-4811, California, Amerika Syarikat

Institut Penyelidikan Virus, Universiti Kyoto, 53 Kawahara, Syogo-in, Sakyo-ku, 606-8397, Kyoto, Jepun

Sankyo Co. Ltd, 2-58 Hiromachi 1-Chome, Shinagawa-Ku, 140-8710, Tokyo, Jepun

Kazuhiko Tamaki & amp Kazuhiko Tanzawa

Pusat Kanser Komprehensif Harold C. Simmons, Pusat Perubatan Barat Daya Universiti Texas, 6000 Harry Hines Boulevard, Dallas, 75235-9111, Texas, Amerika Syarikat


INJEKSI LIQUID SILICONE, 1944 HINGGA 1991

Tentunya sejarah suntikan parafin yang menakutkan seharusnya telah mengajar para doktor dan pesakit untuk berhati-hati terhadap bahan suntikan untuk pembesaran payudara. Namun, pada tahun 1940-an dan 1950-an, banyak doktor dan klinik awam beralih kepada suntikan silikon cair, untuk tujuan ini (9,10). Silikon adalah polimer berangkai silang dimetil siloksan, dengan unit berulang asas:

Pada tahun 1943, Dow Corning Corporation dan Corning Glass membentuk usaha sama di Amerika Syarikat, untuk mengembangkan produk silikon yang akan digunakan untuk tujuan ketenteraan semasa Perang Dunia II. Pada akhirnya, silikon ini digunakan untuk kalis air, untuk menyiapkan minyak dan produk minyak untuk pesawat, untuk melindungi pengubah elektrik, dan untuk menyediakan getah tahan suhu tinggi. Ketika perang berakhir, Dow Corning mengarahkan usaha mereka untuk merumuskan silikon kelas perubatan. Tahap perubatan merujuk kepada bahan yang berkualiti, steril, dan mempunyai kelikatan berterusan. Silikon bertaraf perubatan tidak tersedia sehingga tahun 1960.

Menjelang akhir Perang Dunia II, pelacur di Jepun menggunakan silikon cair kelas industri secara meluas. Anggota tentera Amerika Syarikat lebih suka wanita dengan payudara lebih besar daripada wanita Asia. Tong silikon kelas industri mula hilang secara misterius dari dermaga Jepun, yang ditujukan untuk disuntik ke dalam payudara wanita yang giat ini, untuk memenuhi calon pelanggan mereka.

Di Jepun, silikon cair tersedia dengan nama Elicon (gel silikon tervulkan panas) dan Zeflon (cecair yang dicampurkan dengan garam timah organik). Kedua-dua persiapan ini adalah silikon kelas industri, bukan kelas perubatan. Cecair silikon kelas perubatan dipasarkan oleh syarikat Koken di Jepun, tetapi tidak sampai bertahun-tahun kemudian. Suntikan silikon terus digunakan secara meluas di Jepun dan Asia selepas perang. Sehingga hari ini, ia digunakan di kawasan tertentu di Asia.

Banyak komplikasi suntikan parafin berulang & # x02013 setengah abad kemudian & # x02013 dengan suntikan silikon. Sebilangan dari mereka lebih teruk lagi, kerana kekotoran dan bahan tambahan dalam silikon. Silikon kelas perubatan tidak tersedia sehingga tahun 1960. Sebelum ini, hanya silikon kelas industri yang ada. Bahan ini tidak pernah digunakan untuk suntikan, kerana kekotorannya. Selain itu, dalam banyak persiapan, bahan cemar sengaja ditambahkan ke bahan yang disuntik, seperti dalam formula Sakurai (11). Tujuan mereka adalah untuk menyebabkan reaksi sklerosis pada payudara, mengandung silikon cair dan diharapkan dapat mencegahnya berpindah melalui tisu payudara ke tempat lain. Ejen sclerosing yang biasa termasuk minyak croton, racun kobra, minyak zaitun dan minyak kacang. Kesan buruk cecair yang disuntik sangat serupa dengan parafin. Ini termasuk yang berikut: pemindahan silikon ke bahagian tubuh yang lain, keradangan, perubahan warna, dan pembentukan granuloma, ulserasi dan fistula.

Pada tahun 1960, Dow Corning mengembangkan silikon kelas perubatan pertamanya (12), yang pada awalnya dikenali sebagai & # x02018Dow Corning 200 & # x02019. Bahan ini kemudiannya diperhalusi lebih jauh dan kemudian dikenali sebagai & # x02018Dow Corning 360 & # x02019. Ia dipasarkan dengan nama Dermagen. Tujuan awalnya adalah untuk kalis air pada kulit, terutama pada mangsa luka bakar. Walaupun bahan ini tidak disetujui atau ditujukan untuk suntikan kosmetik pada pasien, bahan ini digunakan secara meluas untuk tujuan ini, oleh doktor tertentu dan oleh klinik awam. Ketika ia digunakan pada payudara, jumlah besar sering digunakan. Sebilangan besar cecair silikon diperoleh dengan alasan palsu bahawa ia digunakan untuk tilam terbakar, untuk merawat kecederaan pada kuda pacuan dan untuk menyiapkan produk baja. Semua penggunaan ini telah diluluskan. Doktor yang tidak bertanggungjawab di Las Vegas menarik sebanyak satu liter bahan dari tong lima galon yang disimpan di pejabat mereka. Ini disuntik ke payudara di bawah tekanan yang besar, menggunakan peralatan yang menyerupai pistol caulking. Dalam perniagaan hiburan, suntikan silikon ini disebut sebagai & # x02018Cleopatra & # x02019s Needle & # x02019. Telah dianggarkan bahawa di Las Vegas pada tahun 1960-an, dua doktor, sendirian, menggunakan silikon untuk menyuntik payudara lebih 10,000 wanita dalam jangka masa 10 tahun (12,13). Tidak ada rekod yang disimpan pada mana-mana pesakit ini.

Menjelang tahun 1965, banyak komplikasi mula muncul dari suntikan silikon cair. Beberapa suntikan telah dilakukan oleh pakar bedah plastik. Namun, banyak yang dilakukan oleh orang awam, yang minimum memenuhi syarat untuk melakukan prosedur seperti ini (9 & # x0201312). Kerana masalah yang disebabkan oleh suntikan silikon yang tidak terkawal oleh pengamal yang tidak berkelayakan, pada tahun 1966, Pentadbiran Makanan dan Dadah (FDA) menetapkan suntikan silikon sebagai & # x02018 ubat baru & # x02019 (14). Keputusan ini menetapkan bahawa silikon harus menjalani penyelidikan makmal tertentu sebelum dapat disetujui untuk digunakan. Sehingga kini, kajian ini tidak pernah dilakukan.

Pada tahun 1966, FDA memberi kuasa kepada sembilan pakar bedah plastik dan seorang pakar dermatologi untuk menyiasat penggunaan kosmetik silikon cair bermutu perubatan Dow Corning & # x02019s (Dow Corning 360) untuk masalah tertentu pada pesakit. Kajian ini hanya terhad kepada doktor tertentu, yang merawat kecacatan wajah tertentu yang tidak dapat dirawat dengan kaedah lain (9). Namun, nampaknya Dow Corning juga memberikan silikon kelas perubatan ini kepada doktor lain, walaupun mereka bukan sebahagian daripada kajian rasmi (15). Selepas itu, Dow Corning menyatakan bahawa kajian asalnya & # x0201 tidak sesempurna yang mungkin & # x0201d. Oleh itu, mereka memutuskan untuk menghentikan usaha untuk mendapatkan persetujuan FDA, kerana mereka tidak dapat & # x0201mencegah penyalahgunaan produk & # x0201d (10). Pada tahun 1975, kerana komplikasi yang mengerikan dari suntikan silikon ke payudara di Las Vegas, negara bagian Nevada menyatakan bahawa itu adalah tindak kejahatan untuk menyuntik silikon atau untuk mengangkut silikon cair ke seluruh negeri.

Pada tahun 1990, lebih daripada 100,000 pesakit telah menerima suntikan gel silikon muka yang asal atau tidak diketahui (13). Pada bulan Ogos 1991, FDA mengeluarkan garis panduan (16) dengan jelas melarang pemasaran atau penjualan silikon cair suntikan untuk tujuan suntikan estetik, sehingga kajian yang sesuai telah selesai. Sehingga kini, penyelidikan klinikal jangka panjang ini tidak pernah dilakukan. Pada tahun 1991, suntikan silikon dilabel sebagai & # x02018dituburkan & # x02019 oleh FDA, untuk menunjukkan bahawa mereka tidak mendapat persetujuan FDA untuk pemasaran atau kajian ilmiah. Pada tahun 1992, FDA mengeluarkan siaran akhbar yang mewajibkan bahawa & # x0201cPakar perubatan tidak lagi dibenarkan menggunakan silikon suntikan untuk rawatan kosmetik melainkan produk tersebut diluluskan oleh FDA untuk kajian pemasaran atau penyelidikan & # x0201d (17). FDA tidak pernah meluluskan penggunaan suntikan silikon cair untuk rawatan kosmetik pesakit, kecuali pada tahun 1966, kajian penyelidikan 10-doktor. Pada tahun 1991/1992, FDA secara rasmi melarang penggunaan semua produk suntikan silikon oleh semua doktor.

Terlepas dari semua amaran dan peringatan, suntikan silikon cair mudah diperoleh oleh mana-mana wanita yang menginginkannya. Mereka sering disuntik di klinik awam di banyak wilayah di Amerika Syarikat. Mereka sangat popular di Mexico dan bahkan di San Francisco. Silikon suntikan juga selalu tersedia secara percuma dari Asia.

Di Kanada, silikon tidak pernah diluluskan untuk tujuan suntikan. Pada tahun 1971, doktor dan lain-lain memperoleh silikon cecair bermutu perubatan secara percuma dari New York, dalam balang 1 lb! Pakar perubatan dan orang awam menggunakan bekalan silikon ini pada tahun 1990-an. Selepas itu, silikon cecair boleh didapati melalui pesanan mel dari Panama, Mexico dan Asia. Beribu-ribu pesakit kemungkinan mendapat suntikan silikon cair di Toronto selama bertahun-tahun.

Di bawah Peraturan Peranti Perubatan Kanada & # x02019s, seperti yang telah dipinda pada tahun 1983, adalah haram untuk menyuntik orang dengan bahan yang belum disetujui oleh pihak berkuasa persekutuan. Ini selalu merangkumi silikon cair. Pada tahun 1992, College of Physicians and Surgeons of Ontario menyatakan bahawa penggunaan suntikan silikon adalah & # x0201cunlawful & # x0201d dan mewakili & # x0201c kesalahan profesional & # x0201d. Walaupun terdapat pernyataan ini, sebilangan doktor Ontario, dan sekurang-kurangnya satu klinik awam di Toronto, terus menyuntik silikon cair selama bertahun-tahun. Sumber silikon suntikan mereka ialah balang 1 lb yang telah diperoleh dari New York pada tahun 1971.

Pada tahun 1994, FDA meluluskan satu bentuk minyak silikon untuk rawatan gangguan yang berkaitan dengan AIDS & # x02013 detasmen retina rumit sekunder daripada retinitis sitomegalovirus (18). Tujuan minyak itu adalah untuk menyediakan & # x0201tamponade retina yang berpanjangan & # x0201d sehingga retina dapat kembali terpasang. Pada tahun 1997, FDA meluluskan formulasi komersial minyak silikon ini, Silikon 1000 (Alcon, USA), untuk rawatan gangguan ini. Pada bulan Mac 2001, FDA membersihkan formulasi komersial lain, Adatosil 5000 (Bausch & # x00026 Lomb, Amerika Syarikat) untuk rawatan bentuk retina terasing ini. Selama beberapa tahun kebelakangan, sebilangan pengamal di Amerika Syarikat telah membeli sediaan silikon cair ini, dengan alasan bahawa ia digunakan untuk rawatan detasmen retina. Namun, mereka telah menyuntik silikon untuk merawat kedutan dan masalah kosmetik lain (12 & # x0201314). Sekiranya detasmen retina, silikon dimaksudkan untuk dikeluarkan setelah pemasangan kembali retina. Penyingkiran ini tidak mungkin dilakukan apabila bahan ini disuntik ke dalam tisu lembut.

FDA tidak pernah meluluskan silikon cair untuk tujuan suntikan kosmetik umum.

Walau bagaimanapun, FDA tidak mempunyai bidang kuasa terhadap amalan perubatan. Oleh itu, doktor sering menggunakan alat yang dibersihkan oleh FDA untuk satu petunjuk, tetapi mereka menggunakannya dalam aplikasi yang sama sekali berbeza atau penggunaan di luar label. Penggunaan luar label ini di luar kuasa FDA. Akta Pemodenan FDA tahun 1997 membenarkan peranti yang dibersihkan oleh FDA digunakan di luar label, untuk sebarang keadaan dalam hubungan doktor-pesakit (18).

Pada masa ini, ujian klinikal yang diluluskan oleh FDA dilaporkan sedang berjalan untuk produk silikon cair khusus untuk rawatan lipoatrofi wajah yang berkaitan dengan HIV dan untuk digunakan dalam petunjuk kosmetik (14,18). Tidak banyak yang diketahui mengenai kajian ini kecuali ia melibatkan penggunaan teknik tusukan bersiri microdroplet, seperti yang dijelaskan oleh Orentreich (19). Teknik suntikan ini dilaporkan pada akhir 1970-an. Ini terdiri daripada memasukkan titisan silikon cair 0,01 mL atau lebih kecil ke dalam tisu subdermal pada selang 2 mm hingga 10 mm. Rawatan dijarakkan sekurang-kurangnya satu bulan sehingga hasil yang diinginkan diperoleh. Suntikan mikrodroplet ini terbukti dapat menghasilkan reaksi keradangan ringan, yang mengakibatkan tindak balas fibroblastik. Fibrosis yang dihasilkan bertanggungjawab untuk peningkatan tisu lembut. Sebaliknya, suntikan dengan dos yang lebih besar (lebih besar daripada 0.05 mL) telah terbukti menghasilkan granuloma dan reaksi badan asing. Dalam kajian ini, pesakit akan dinilai sekurang-kurangnya tujuh tahun. Sehingga kini, belum ada kajian susulan yang dilaporkan.

Mamografi payudara yang disuntik dengan silikon menunjukkan salah satu daripada dua corak: jisim sista berkisar antara 0,2 cm hingga 2,0 cm, selalunya dengan kalsifikasi (Gambar 3A), atau kawasan kelegapan yang besar jika jumlah yang lebih besar telah disuntik ke kawasan payudara ( Rajah 3B).


Kandungan

Pada manusia, tisu adiposa terletak: di bawah kulit (lemak subkutan), di sekitar organ dalaman (lemak viseral), di sumsum tulang (sumsum tulang kuning), intermuskular (Sistem otot) dan di payudara (tisu payudara). Tisu adiposa terdapat di lokasi tertentu, yang disebut sebagai depot adipose. Selain daripada adiposit, yang merangkumi peratusan sel tertinggi dalam tisu adiposa, jenis sel lain ada, yang secara kolektif disebut fraksi vaskular stromal (SVF) sel. SVF merangkumi preadiposit, fibroblas, makrofag tisu adiposa, dan sel endotel.

Tisu adiposa mengandungi banyak saluran darah kecil. Dalam sistem integumen, yang merangkumi kulit, ia terkumpul di tahap terdalam, lapisan subkutan, memberikan penebat dari panas dan sejuk. Di sekitar organ, ia menyediakan pelindung. Namun, fungsi utamanya adalah menjadi simpanan lipid, yang dapat dioksidakan untuk memenuhi keperluan tenaga tubuh dan melindunginya dari glukosa berlebihan dengan menyimpan trigliserida yang dihasilkan oleh hati dari gula, walaupun beberapa bukti menunjukkan bahawa kebanyakan sintesis lipid dari karbohidrat berlaku di tisu adiposa itu sendiri. [4] Depot adiposa di bahagian tubuh yang berlainan mempunyai profil biokimia yang berbeza. Dalam keadaan normal, ia memberi maklum balas untuk kelaparan dan diet ke otak.

Edit Tikus

Tikus mempunyai lapan depot adiposa utama, empat daripadanya berada di dalam rongga perut. [1] Depot gonad berpasangan melekat pada rahim dan ovari pada wanita dan epididimis dan testis pada lelaki depot retroperitoneal berpasangan dijumpai di sepanjang dinding punggung perut, mengelilingi buah pinggang, dan, apabila besar, memanjang ke pelvis . Depot mesenterik membentuk jaring seperti lem yang menyokong usus dan depot omental (yang berasal berhampiran perut dan limpa) dan - apabila besar - meluas ke perut ventral. Kedua-dua depot mesenterik dan omental menggabungkan banyak tisu limfoid sebagai kelenjar getah bening dan bintik susu.

Dua depot dangkal adalah depot inguinal berpasangan, yang terdapat di anterior ke bahagian atas anggota belakang (di bawah kulit) dan depot subskapular, campuran medial tisu adiposa coklat yang berdekatan dengan kawasan tisu adiposa putih, yang dijumpai di bawah kulit antara puncak dorsal dari scapula. Lapisan tisu adiposa coklat di depot ini sering diliputi oleh "frosting" tisu adiposa putih yang kadang-kadang kedua-dua jenis lemak ini (coklat dan putih) sukar dibezakan. Depot inguinal merangkumi kumpulan inguinal kelenjar getah bening. Depot kecil termasuk perikardial, yang mengelilingi jantung, dan depot popliteal berpasangan, antara otot utama di belakang lutut, masing-masing mengandungi satu kelenjar getah bening yang besar. [5] Dari semua depot tetikus, depot gonad adalah yang terbesar dan paling mudah dibedah, [6] yang terdiri daripada kira-kira 30% lemak yang dapat dibedah. [7]

Suntingan Obesiti

Pada orang yang gemuk, lebihan tisu adiposa yang tergantung ke bawah dari perut disebut sebagai panniculus. Panniculus merumitkan pembedahan individu yang gemuk. Ini mungkin kekal sebagai "celemek kulit" secara harfiah jika seseorang yang mengalami kegemukan dengan cepat kehilangan sejumlah besar lemak (hasil biasa dari pembedahan pintasan gastrik). Obesiti dirawat melalui senaman, diet, dan terapi tingkah laku. Pembedahan rekonstruktif adalah salah satu kaedah rawatan. [8]

Lemak viseral

Lemak viseral atau lemak perut [9] (juga dikenali sebagai lemak organ atau lemak intra-perut) terletak di dalam rongga perut, dibungkus di antara organ (perut, hati, usus, ginjal, dll.). Lemak viseral berbeza dengan lemak subkutan di bawah kulit, dan lemak intramuskular diselingi pada otot rangka. Lemak di bahagian bawah badan, seperti di paha dan punggung, adalah subkutan dan tisu tidak dijarakkan secara konsisten, sedangkan lemak di perut kebanyakannya bersifat visceral dan separa cairan. [10] Lemak viseral terdiri daripada beberapa depot adiposa, termasuk mesenterik, tisu adiposa putih epididimis (EWAT), dan depot perirenal. Lemak viseral sering dinyatakan dari segi luasnya dalam cm 2 (VFA, kawasan lemak viseral). [11]

Kelebihan lemak viseral dikenali sebagai obesiti pusat, atau "lemak perut", di mana perut menonjol secara berlebihan. Perkembangan baru seperti Body Volume Index (BVI) dirancang khusus untuk mengukur jumlah perut dan lemak perut. Lemak viseral yang berlebihan juga dikaitkan dengan diabetes jenis 2, [12] ketahanan insulin, [13] penyakit radang, [14] dan penyakit lain yang berkaitan dengan obesiti. [15] Begitu juga, pengumpulan lemak leher (atau tisu adiposa serviks) telah terbukti berkaitan dengan kematian. [16] Beberapa kajian menunjukkan bahawa lemak viseral dapat diramalkan dari ukuran antropometri sederhana, [17] dan meramalkan kematian dengan lebih tepat daripada indeks jisim badan atau lilitan pinggang. [18]

Lelaki lebih cenderung menyimpan lemak di perut kerana perbezaan hormon seks. Hormon seks wanita menyebabkan lemak disimpan di punggung, paha, dan pinggul pada wanita. [19] [20] Ketika wanita mencapai menopaus dan estrogen yang dihasilkan oleh ovari menurun, lemak berpindah dari punggung, pinggul dan paha ke pinggang [21] kemudian lemak disimpan di perut. [10]

Latihan dengan intensiti tinggi adalah salah satu cara untuk mengurangkan lemak perut secara berkesan. [22] [23] Satu kajian menunjukkan sekurang-kurangnya 10 MET-jam seminggu latihan aerobik diperlukan untuk pengurangan lemak viseral. [24] Diet tenaga yang digabungkan dengan senaman akan mengurangkan jumlah lemak badan dan nisbah tisu adiposa visceral ke tisu adiposa subkutan, menunjukkan mobilisasi yang lebih baik untuk lemak viseral berbanding lemak subkutan. [25]

Lemak epikardial

Tisu epicardial adipose (EAT) adalah bentuk lemak viseral tertentu yang tersimpan di sekitar jantung dan didapati organ aktif metabolik yang menghasilkan pelbagai molekul bioaktif, yang mungkin mempengaruhi fungsi jantung dengan ketara. [26] Perbezaan komponen yang ketara telah diperhatikan dalam membandingkan MAKAN dengan lemak subkutan, menunjukkan kesan depot khas asam lemak tersimpan pada fungsi dan metabolisme adiposit. [27]

Lemak subkutan

Sebilangan besar lemak nonvisceral yang tersisa dijumpai tepat di bawah kulit di kawasan yang disebut hypodermis. [28] Lemak subkutan ini tidak berkaitan dengan banyak patologi klasik yang berkaitan dengan obesiti, seperti penyakit jantung, barah, dan strok, dan beberapa bukti bahkan menunjukkan bahawa ia mungkin bersifat pelindung. [29] Corak taburan lemak badan (atau ginekoid) yang biasanya wanita di sekitar pinggul, paha, dan punggung adalah lemak subkutan, dan oleh itu menimbulkan risiko kesihatan yang lebih rendah berbanding lemak viseral. [30] [31]

Seperti organ lemak lain, lemak subkutan adalah bahagian aktif sistem endokrin, merembeskan hormon leptin dan resistin. [28]

Hubungan antara lapisan adiposa subkutan dan jumlah lemak badan pada seseorang sering dimodelkan dengan menggunakan persamaan regresi. Persamaan yang paling popular dibentuk oleh Durnin dan Wormersley, yang dengan teliti menguji banyak jenis lipatan kulit, dan, sebagai hasilnya, membuat dua formula untuk mengira ketumpatan badan lelaki dan wanita. Persamaan ini memperlihatkan korelasi terbalik antara lipatan kulit dan ketumpatan badan — ketika jumlah lipatan kulit meningkat, kepadatan badan menurun. [32]

Faktor-faktor seperti jantina, umur, ukuran populasi atau pemboleh ubah lain boleh menjadikan persamaan itu tidak sah dan tidak dapat digunakan, dan, pada 2012 [kemas kini], persamaan Durnin dan Wormersley hanya menjadi anggaran tahap kegemukan seseorang. Formula baru masih dibuat. [32]

Lemak sumsum

Lemak sumsum, juga dikenali sebagai tisu adiposa sumsum (MAT), adalah depot adiposa yang kurang difahami yang berada di dalam tulang dan diselingi dengan sel-sel hematopoietik serta unsur-unsur tulang. Adiposit di depot ini berasal dari sel stem mesenchymal (MSC) yang dapat menimbulkan sel lemak, sel tulang dan juga jenis sel lain. Fakta bahawa MAT meningkat dalam pengaturan pembatasan kalori / anoreksia adalah ciri yang membezakan depot ini dari depot lemak lain. [33] [34] [35] Latihan mengatur MAT, menurunkan kuantiti MAT dan mengurangkan ukuran adiposit sumsum. [36] [37] [38] Peraturan latihan lemak sumsum menunjukkan bahawa ia mempunyai persamaan fisiologi dengan depot adiposa putih yang lain. Lebih-lebih lagi, peningkatan MAT dalam obesiti menunjukkan persamaan dengan depot lemak putih. [36]

Lemak ektopik

Lemak ektopik adalah penyimpanan trigliserida dalam tisu selain tisu adiposa, yang seharusnya hanya mengandung sejumlah kecil lemak, seperti hati, otot rangka, jantung, dan pankreas. [1] Ini boleh mengganggu fungsi sel dan oleh itu fungsi organ dan dikaitkan dengan ketahanan insulin pada diabetes jenis-2. [39] Ia disimpan dalam jumlah yang agak tinggi di sekitar organ rongga perut, tetapi tidak boleh dikelirukan dengan lemak viseral.

Punca khusus untuk pengumpulan lemak ektopik tidak diketahui. Penyebabnya kemungkinan merupakan gabungan faktor genetik, persekitaran, dan tingkah laku yang terlibat dalam pengambilan tenaga berlebihan dan penurunan aktiviti fizikal. Penurunan berat badan yang ketara dapat mengurangkan simpanan lemak ektopik di semua organ dan ini dikaitkan dengan peningkatan fungsi organ tersebut. [39]

Dalam kes terakhir, intervensi penurunan berat badan yang tidak invasif seperti diet atau senaman dapat menurunkan lemak ektopik (terutama pada jantung dan hati) pada kanak-kanak dan orang dewasa yang berlebihan berat badan atau gemuk. [40] [41]

Asid lemak bebas (FFA) dibebaskan dari lipoprotein oleh lipoprotein lipase (LPL) dan memasuki adiposit, di mana mereka disatukan kembali menjadi trigliserida dengan mengestrastikannya ke gliserol. Tisu lemak manusia mengandungi sekitar 87% lipid. [42]

Terdapat aliran berterusan FFA memasuki dan meninggalkan tisu adiposa. Arah bersih fluks ini dikendalikan oleh insulin dan leptin - jika insulin meningkat, maka terdapat aliran masuk FFA bersih, dan hanya apabila insulin rendah FFA dapat meninggalkan tisu adiposa. Rembesan insulin dirangsang oleh gula darah tinggi, yang disebabkan oleh pengambilan karbohidrat. [43]

Pada manusia, lipolisis (hidrolisis trigliserida menjadi asid lemak bebas) dikendalikan melalui kawalan seimbang reseptor B-adrenergik lipolitik dan antilipolisis a2A-adrenergik yang dimediasi oleh reseptor.

Sel-sel lemak mempunyai peranan fisiologi yang penting dalam menjaga kadar trigliserida dan asid lemak bebas, serta menentukan daya tahan insulin. Lemak perut mempunyai profil metabolik yang berbeza-lebih cenderung menyebabkan ketahanan insulin. Ini menjelaskan secara besar-besaran mengapa obesiti pusat adalah penanda toleransi glukosa yang terganggu dan merupakan faktor risiko bebas untuk penyakit kardiovaskular (walaupun tidak terdapat diabetes mellitus dan hipertensi). [44] Kajian monyet betina di Wake Forest University (2009) mendapati bahawa individu yang menderita tekanan tinggi mempunyai tahap lemak viseral yang lebih tinggi di dalam badan mereka. Ini menunjukkan kemungkinan hubungan sebab-akibat antara keduanya, di mana tekanan mendorong pengumpulan lemak viseral, yang seterusnya menyebabkan perubahan hormon dan metabolik yang menyumbang kepada penyakit jantung dan masalah kesihatan yang lain. [45]

Kemajuan terkini dalam bioteknologi memungkinkan pengambilan sel stem dewasa dari tisu adiposa, yang memungkinkan rangsangan pertumbuhan semula tisu menggunakan sel pesakit sendiri. Sebagai tambahan, sel induk yang berasal dari adiposa dari manusia dan hewan dilaporkan dapat diprogram ulang secara efisien menjadi sel induk pluripoten yang diinduksi tanpa memerlukan sel pengumpan. [46] Penggunaan sel pesakit sendiri mengurangkan kemungkinan penolakan tisu dan mengelakkan masalah etika yang berkaitan dengan penggunaan sel induk embrio manusia. [47] Banyak bukti yang menunjukkan bahawa depot lemak yang berbeza (iaitu perut, omental, perikardial) menghasilkan sel stem yang berasal dari adiposa dengan ciri yang berbeza. [47] [48] Ciri-ciri yang bergantung pada depot ini meliputi kadar percambahan, imunofenotip, potensi pembezaan, ekspresi gen, serta kepekaan terhadap keadaan kultur hipoksia. [49] Tahap oksigen sepertinya memainkan peranan penting pada metabolisme dan secara umum fungsi sel induk yang berasal dari adiposa. [50]

Adipose tissue is a major peripheral source of aromatase in both males and females, contributing to the production of estradiol. [51]

Adipose tissues also secrete a type of cytokines (cell-to-cell signalling proteins) called adipokines (adipose cytokines), which play a role in obesity-associated complications. Perivascular adipose tissue releases adipokines such as adiponectin that affect the contractile function of the vessels that they surround. [1] [52]

Brown fat Edit

Brown fat or brown adipose tissue (BAT) is a specialized form of adipose tissue important for adaptive thermogenesis in humans and other mammals. BAT can generate heat by "uncoupling" the respiratory chain of oxidative phosphorylation within mitochondria through tissue-specific expression of uncoupling protein 1 (UCP1). [53] BAT is primarily located around the neck and large blood vessels of the thorax, where it may effectively act in heat exchange. BAT is robustly activated upon cold exposure by the release of catecholamines from sympathetic nerves that results in UCP1 activation. BAT activation may also occur in response to overfeeding. [54] UCP1 activity is stimulated by long chain fatty acids that are produced subsequent to β-adrenergic receptor activation. [53] UCP1 is proposed to function as a fatty acid proton symporter, although the exact mechanism has yet to be elucidated. [55] In contrast, UCP1 is inhibited by ATP, ADP, and GTP. [56]

Attempts to simulate this process pharmacologically have so far been unsuccessful. Techniques to manipulate the differentiation of "brown fat" could become a mechanism for weight loss therapy in the future, encouraging the growth of tissue with this specialized metabolism without inducing it in other organs. A review on the eventual therapeutic targeting of brown fat to treat human obesity was published by Samuelson and Vidal-Puig in 2020. [57]

Until recently, brown adipose tissue was thought to be primarily limited to infants in humans, but new evidence has now overturned that belief. Metabolically active tissue with temperature responses similar to brown adipose was first reported in the neck and trunk of some human adults in 2007, [58] and the presence of brown adipose in human adults was later verified histologically in the same anatomical regions. [59] [60] [61]

Beige fat and WAT browning Edit

Browning of WAT, also referred to as "beiging", occurs when adipocytes within WAT depots develop features of BAT. Beige adipocytes take on a multilocular appearance (containing several lipid droplets) and increase expression of uncoupling protein 1 (UCP1). [62] In doing so, these normally energy-storing adipocytes become energy-releasing adipocytes.

The calorie-burning capacity of brown and beige fat has been extensively studied as research efforts focus on therapies targeted to treat obesity and diabetes. The drug 2,4-dinitrophenol, which also acts as a chemical uncoupler similarly to UCP1, was used for weight loss in the 1930s. However, it was quickly discontinued when excessive dosing led to adverse side effects including hyperthermia and death. [62] β3 agonists, like CL316,243, have also been developed and tested in humans. However, the use of such drugs has proven largely unsuccessful due to several challenges, including varying species receptor specificity and poor oral bioavailability. [63]

Cold is a primary regulator of BAT processes and induces WAT browning. Browning in response to chronic cold exposure has been well documented and is a reversible process. A study in mice demonstrated that cold-induced browning can be completely reversed in 21 days, with measurable decreases in UCP1 seen within a 24-hour period. [64] A study by Rosenwald et al. revealed that when the animals are re-exposed to a cold environment, the same adipocytes will adopt a beige phenotype, suggesting that beige adipocytes are retained. [65]

Transcriptional regulators, as well as a growing number of other factors, regulate the induction of beige fat. Four regulators of transcription are central to WAT browning and serve as targets for many of the molecules known to influence this process. [66] These include peroxisome proliferator-activated receptor gamma (PPARγ), PR domain containing 16 (PRDM16), [67] peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), and Early B-Cell Factor-2 (EBF2). [68] [69] [70]

The list of molecules that influence browning has grown in direct proportion to the popularity of this topic and is constantly evolving as more knowledge is acquired. Among these molecules are irisin and fibroblast growth factor 21 (FGF21), which have been well-studied and are believed to be important regulators of browning. Irisin is secreted from muscle in response to exercise and has been shown to increase browning by acting on beige preadipocytes. [71] FGF21, a hormone secreted mainly by the liver, has garnered a great deal of interest after being identified as a potent stimulator of glucose uptake and a browning regulator through its effects on PGC-1α. [62] It is increased in BAT during cold exposure and is thought to aid in resistance to diet-induced obesity [72] FGF21 may also be secreted in response to exercise and a low protein diet, although the latter has not been thoroughly investigated. [73] [74] Data from these studies suggest that environmental factors like diet and exercise may be important mediators of browning. In mice, it was found that beiging can occur through the production of methionine-enkephalin peptides by type 2 innate lymphoid cells in response to interleukin 33. [75]

Genomics and bioinformatics tools to study browning Edit

Due to the complex nature of adipose tissue and a growing list of browning regulatory molecules, great potential exists for the use of bioinformatics tools to improve study within this field. Studies of WAT browning have greatly benefited from advances in these techniques, as beige fat is rapidly gaining popularity as a therapeutic target for the treatment of obesity and diabetes.

DNA microarray is a bioinformatics tool used to quantify expression levels of various genes simultaneously, and has been used extensively in the study of adipose tissue. One such study used microarray analysis in conjunction with Ingenuity IPA software to look at changes in WAT and BAT gene expression when mice were exposed to temperatures of 28 and 6 °C. [76] The most significantly up- and downregulated genes were then identified and used for analysis of differentially expressed pathways. It was discovered that many of the pathways upregulated in WAT after cold exposure are also highly expressed in BAT, such as oxidative phosphorylation, fatty acid metabolism, and pyruvate metabolism. [76] This suggests that some of the adipocytes switched to a beige phenotype at 6 °C. Mössenböck et al. also used microarray analysis to demonstrate that insulin deficiency inhibits the differentiation of beige adipocytes but does not disturb their capacity for browning. [77] These two studies demonstrate the potential for the use of microarray in the study of WAT browning.

RNA sequencing (RNA-Seq) is a powerful computational tool that allows for the quantification of RNA expression for all genes within a sample. Incorporating RNA-Seq into browning studies is of great value, as it offers better specificity, sensitivity, and a more comprehensive overview of gene expression than other methods. RNA-Seq has been used in both human and mouse studies in an attempt characterize beige adipocytes according to their gene expression profiles and to identify potential therapeutic molecules that may induce the beige phenotype. One such study used RNA-Seq to compare gene expression profiles of WAT from wild-type (WT) mice and those overexpressing Early B-Cell Factor-2 (EBF2). WAT from the transgenic animals exhibited a brown fat gene program and had decreased WAT specific gene expression compared to the WT mice. [78] Thus, EBF2 has been identified as a potential therapeutic molecule to induce beiging.

Chromatin immunoprecipitation with sequencing (ChIP-seq) is a method used to identify protein binding sites on DNA and assess histone modifications. This tool has enabled examination of epigenetic regulation of browning and helps elucidate the mechanisms by which protein-DNA interactions stimulate the differentiation of beige adipocytes. Studies observing the chromatin landscapes of beige adipocytes have found that adipogenesis of these cells results from the formation of cell specific chromatin landscapes, which regulate the transcriptional program and, ultimately, control differentiation. Using ChIP-seq in conjunction with other tools, recent studies have identified over 30 transcriptional and epigenetic factors that influence beige adipocyte development. [78]

Genetics Edit

The thrifty gene hypothesis (also called the famine hypothesis) states that in some populations the body would be more efficient at retaining fat in times of plenty, thereby endowing greater resistance to starvation in times of food scarcity. This hypothesis, originally advanced in the context of glucose metabolism and insulin resistance, has been discredited by physical anthropologists, physiologists, and the original proponent of the idea himself with respect to that context, although according to its developer it remains "as viable as when [it was] first advanced" in other contexts. [79] [80]

In 1995, Jeffrey Friedman, in his residency at the Rockefeller University, together with Rudolph Leibel, Douglas Coleman et al. discovered the protein leptin that the genetically obese mouse lacked. [81] [82] [83] Leptin is produced in the white adipose tissue and signals to the hypothalamus. When leptin levels drop, the body interprets this as a loss of energy, and hunger increases. Mice lacking this protein eat until they are four times their normal size.

Leptin, however, plays a different role in diet-induced obesity in rodents and humans. Because adipocytes produce leptin, leptin levels are elevated in the obese. However, hunger remains, and—when leptin levels drop due to weight loss—hunger increases. The drop of leptin is better viewed as a starvation signal than the rise of leptin as a satiety signal. [84] However, elevated leptin in obesity is known as leptin resistance. The changes that occur in the hypothalamus to result in leptin resistance in obesity are currently the focus of obesity research. [85]

Gene defects in the leptin gene (ob) are rare in human obesity. [86] As of July 2010 [update] , only 14 individuals from five families have been identified worldwide who carry a mutated ob gene (one of which was the first ever identified cause of genetic obesity in humans)—two families of Pakistani origin living in the UK, one family living in Turkey, one in Egypt, and one in Austria [87] [88] [89] [90] [91] —and two other families have been found that carry a mutated ob receptor. [92] [93] Others have been identified as genetically partially deficient in leptin, and, in these individuals, leptin levels on the low end of the normal range can predict obesity. [94]

Several mutations of genes involving the melanocortins (used in brain signaling associated with appetite) and their receptors have also been identified as causing obesity in a larger portion of the population than leptin mutations. [95]

Physical properties Edit

Adipose tissue has a density of

0.9 g/ml. [96] Thus, a person with more adipose tissue will float more easily than a person of the same weight with more muscular tissue, since muscular tissue has a density of 1.06 g/ml. [97]

A body fat meter is a tool used to measure the body fat to weight ratio in the human body. Different meters use various methods to determine the ratio. They tend to under-read body fat percentage.

In contrast with clinical tools, one relatively inexpensive type of body fat meter uses the principle of bioelectrical impedance analysis (BIA) in order to determine an individual's body fat percentage. To achieve this, the meter passes a small, harmless, electric current through the body and measures the resistance, then uses information on the person's weight, height, age, and sex to calculate an approximate value for the person's body fat percentage. The calculation measures the total volume of water in the body (lean tissue and muscle contain a higher percentage of water than fat), and estimates the percentage of fat based on this information. The result can fluctuate several percentage points depending on what has been eaten and how much water has been drunk before the analysis.

Before bioelectrical impedance analysis machines were developed, there were many different ways in analyzing body composition such as skin fold methods using calipers, underwater weighing, whole body air displacement plethysmography (ADP) and DXA.

Within the fat (adipose) tissue of CCR2 deficient mice, there is an increased number of eosinophils, greater alternative Macrophage activation, and a propensity towards type 2 cytokine expression. Furthermore, this effect was exaggerated when the mice became obese from a high fat diet. [98]


Latar belakang

Massively parallel sequencing by next generation sequencing (NGS) technology has been rapidly introduced into basic and translational research in oncology, due to the ability of identifying the complete landscape of genetic alterations in many tumor types [1–5].

Most genotyping studies have been performed using fresh frozen (FF) tissues, and have provided great insights into the cancer molecular biology. However, the higher quality of DNA extracted from FF tissue is offset by the reduced availability of the samples, which does not allow to perform large-scale retrospective studies. Therefore in the recent years, many efforts have been addressed to set up strategies to apply massively parallel sequencing technology to formalin-fixed, paraffin-embedded (FFPE) specimens. While FFPE specimens are now frequently analyzed by amplicon-based or targeted-capture NGS panels [6–10], the possibility to reliably perform whole genome or whole exome sequencing (WES) in archival tumor samples still represents a challenge, both from the technical and bioinformatic point of view [11–16].

Gastrointestinal stromal tumors (GIST) are mesenchymal tumors that most frequently arise in the gastrointestinal tract. GIST are characterized by mutually exclusive KIT (85 %) or platelet-derived growth factor receptor alpha (PDGFRA) (5-10 %) gain of function mutations, leading to constitutive ligand-independent activation of receptor signalling [17–19]. The knowledge about the oncogenic mechanisms responsible for GIST onset paved the way for the effective introduction of tyrosine-kinase inhibitors (TKIs) in the standard treatment protocols and the recognition of the clinical impact and predictive significance of molecularly-defined subtypes [20, 21]. Up to now, about 10-15 % of GIST do not exhibit neither KIT or PDGFRA mutations and have been defined as KIT/PDGFRA wild type (WT), which represent an extremely heterogeneous subgroup, characterized by different subsets with distinct molecular hallmarks [22, 23].

In this complex scenario, in which the molecular biology plays a certain relevant role, but the FF specimens are often not available, the feasibility of high-throughput genomic studies on FFPE tissue would allow to perform larger prospective and retrospective studies on all these small subsets of GIST, expanding the reproducibility and the reliability of the data.

This study is aimed to develop a reliable approach to perform WES on archival tumor samples from GIST patients, in order to evaluate how data generated from FFPE material can be generated and potentially used in routine clinical settings. Herein we reported the first pivotal study on the comparison between data obtained by whole exome analysis on four FFPE and FF GIST samples, showing an high degree of concordance for all the variants found, including common polymorphism and novel somatic variants.


PREPARATION OF THE DILUTION SERIES

Dilute the labeling probe using dilution buffer to a starting concentration of 2.5 pmol/μl.

Make a dilution series in Eppendorf tubes of purified probe to give nucleic acid concentrations of 300 pg/μl, 100 pg/μl, 30 pg/μl, 10 pg/μl, 3 pg/μl, and one tube containing diluent only. Ensure that all tube volumes are equal. Repeat the same dilution series with your control or used pre-labeled (with control) test strips.

Apply 1 μl drops from each tube onto the nylon membrane (Roche). The control dilutions should be lined up with the test sample dilution concentration. For an example of placing spots, see Fig. 26.2 .

Label the position of each application with a pencil on the side of the strip (not on the strip).

Fix the nucleic acid to the membrane by either baking the membrane for 30 min at 120ଌ or using a UV light.

Wash the membrane briefly in washing buffer.

Immerse in blocking solution for 10 min.

Incubate with reagents used in ISH detection technique.

Dilute reagents in blocking solution and use this solution for washing.

Detect enzyme using the same solutions and procedure as for ISH method.

Rinse in double distilled water and blot dry.


Case presentation

Case details

An eight-month-old warmblood stallion was presented for examination due to ataxia manifested by problems with coordination, stumbling and spontaneous falling. The symptoms were first observed at the pasture when the horse grazed with other colts. There were no direct signs of any trauma at pasture. There was no history of ataxic horse in the family, the horse was raised in one location and was not transported. It did not have contact with unknown horses/ animals or sport horses.

Clinical and diagnostic findings

The horse underwent a clinical examination consisting of observation, palpation and auscultation. The horse did not present any signs of pain. No nasal discharge or coughing, oedema or wounds/scars has been detected. Urination, defecation and feed uptake were normal. Body temperature measurement, heart and lung auscultation including respiratory and heart rates were in physiological values. Mucous membranes were pink with CRT < 2 s. Palpation of the lymph nodes did not reveal any abnormalities. Both testes has been palpable in the scrotum. Ophthalmologic examination showed no abnormalities. There were no other clinical signs of infectious diseases. Laboratory blood analysis, which included a complete blood count and serum chemistry as well as micro- and macro-element and vitamin E analysis was performed and did not reveal any abnormalities. The orthopedic examination revealed gait deficits, graded as 3–4/5 according to the Mayhew scale [21, 23]. The neurological examination revealed no changes in the mental status of the horse. There were no cranial nerve deficits and all spinal reflexes were normal [14, 20]. The patient presented hypermetria during walk and trot. While resting in the box, the horse tried to find a stable, comfortable position and moved only when necessary. The horse also had problems with turning and kept its inner hind leg on the ground. Additionally, the horse could not be walked backward there was risk of loss of balance and falling. Based on the neurological examination, the lesion was localised to the cervical spinal cord [14, 20]. It was therefore decided to perform standing lateral radiographs of the cervical vertebrae, which did not reveal any malformation or malallineation changes. The differential diagnoses included CVSM, aberrant parasite migration and equine degenerative myeloencephalopathy (EDM). Equine protozoal meningitis (EPM), due to geographic location, was excluded [7, 11, 28]. The radiographs were evaluated for the measurements of the cervical inter- and intravertebral ratio (Table. 1). The lowest sagittal intravertebral ratio was noted at the C3/C4 level and amounted to 31.69% with 55.06% sagittal intervertebral ratio, while the values obtained at the C4/C5 level amounted to 47.77 and 50.54%, respectively. It was decided not to perform a CSF tap following an analysis of the radiograms.

Due to financial constraints, conservative therapy was attempted, and included an administration of nonsteroidal anti-inflammatory drugs (flunixin meglumin 1.1 mg/kg BW q24h), Footnote 1 a high dose of vitamin E Footnote 2 (10,000 IU/day) and exercise restriction. A control examination performed after a month of treatment did not reveal any improvement in the horse’s clinical findings. At that stage, it was clear that the horse would not be fit for sport use and the owner did not agree to further examinations. Due to the high degree of ataxia of the horse, which posed a threat to the animal itself as well as its caregivers and no possibility of effective treatment, the animal was euthanised. General anaesthesia was induced by diazepam Footnote 3 (0.02 mg/kg IV) and ketamine Footnote 4 (2.2 mg/kg IV), after achieving sufficient sedation with xylazine Footnote 5 (1.1 mg/kg IV), and euthanasia performed by administration of pentobarbital sodium Footnote 6 (140 mg/kg IV after receiving sufficient anaesthesia).

Post-mortem evaluation

Immediately after euthanasia and after obtaining the owner consent, myelography was performed to confirm the diagnosis. The horse was placed in right lateral recumbency. A non-ionic contrast medium (Accupaque® 350 Footnote 7 10 ml/100 kg iohexol 350 mg I/ml e ) was slowly injected subarachnoidally (Spine- Ject®, 18G, 3 1/2″) into the atlanto-occipital space after removing an equal quantity of cerebrospinal fluid. The diagnostic procedure consisted of radiographs in neutral, flexed, and extended positions of the neck. The dorsal myelographic column (DMC) and the dural diameter (DD) were measured based on the myelographic dye column. The measurements of the myelographic dye column revealed a 67% reduction of the dorsal myelographic column (DMC) and a 64% decrease in the dural diameter (DD) at the level of C3/C4 (Fig.1A, B, Tab.1) in a flexed spine position, which confirmed type I CVSM (a dynamic lesion). Next, an anatomical dissection was performed. The cervical vertebral column was removed and immersed in a tank of neutral buffered formalin for 1 week, than sectioned saggitally [33]. The cervical vertebrae and the vertebral canal were macroscopically inspected to obtain actual measurements at the stenotic site. Real scenario measurements of the vertebral canal were taken during the anatomical dissection and are summarised in Table 2 (Fig. 2). According to the measurements, there was a 44% narrowing of the vertebral canal at C3/C4 level and 27% narrowing of the vertebral canal at C4/C5 level respectively. The spinal cord was collected as fast as possible after euthanasia. Following fixation, the segments of the C3/C4 spinal cord compression were sectioned transversely and divided into several parts.

A- Lateral myelogram projection presenting the C3-C5 cervical vertebrae in a flexed position showing a significant reduction of the dorsal dye column at the level of the C3/C4 articulation indicating spinal canal narrowing and suggesting compression of the spinal cord. The reduction of the ventral dye column is typical for cervical flexion radiographs. The dural diameter and dorsal myelographic column measurements are marked. B- Lateral myelogram projection presenting the C2-C4 cervical vertebrae in a neutral position of the neck. No degenerative joint disease or malformation changes were noticed at the level of C3/C4

Anatomical dissection with longitudinal cross section of the cervical spine presenting the C3-C5 region

Histopathologic and morphologic examination

All the samples were fixed in 4% buffered formalin and embedded in paraffin blocks. Five μm paraffin sections were obtained using a rotary microtome, and they were stained with hematoxylin and eosin (HE) and Masson trichrome (MTC) according to the relevant histological protocols. Section analysis was carried out with an optical microscope (Axio Imager A1 Carl Zeiss).

Imunohistokimia

CD68/KP1

For immunohistochemistry analysis, the paraffin embedded tissue was cut into 4 μm thick section, placed on silanized slides (Dako, S 3003) and dried for 12 h in an incubator at 37 °C. The dried sections were then dehydrated and they were pre-treated with 0.01 M citrate buffer solution at a higher pH in an incubator at 97 °C for 20 min to unmask the antigenic sites. The sections were rinsed in 0.01% phosphate buffered saline (PBS). Then, they were treated with enzyme block FLEX peroxidase for 5 min, rinsed in PBS and subsequently overlaid by the primary antibody CD68/KP1 (Dako). Next, the slides were rinsed in PBS, labelled using a Polymer Flex/HRP for 20 min, rinsed in PBS for 5 min and covered by Chromogen Flex DAB with Subchromogen for 10 min. After rinsing the slides in PBS, they were counterstained using FLEX Hematoxylin for 5 min, washed with deionized water and PBS and analysed with an optical microscope (Zeiss Axio Scope A1 Carl Zeiss). A positive and a negative control were performed for each sample.

Immunostaining was performed using a standard technique, according to protocols designed at the Department of Experimental Biology, Faculty of Biology and Animal Science at the Wroclaw University of Environmental and Life Sciences [2]. The tissue samples were cut into 3 μm-thick sections, deparaffinised in xylene and washed in a series of decreasing alcohol concentrations from 100 to 50%. An EnVision System (Dako) was used to visualize the antigen-antibody reaction. Immunoperoxidase labelling was performed using polyclonal antibodies against TNF-α (R&D Systems, USA). Antigen heat-induced retrieval was performed by incubating the slides with a target retrieval solution (pH 9.0 Dako) for 20 min at 96 °C. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide, and the tissue sections were then washed with Tris-buffered saline (TBS) for 5 min at room temperature. Next, the slides were labelled with primary antibodies for 20 min at 20 °C. The antibodies were diluted to 1:10. Then the sections were counterstained with Mayer’s hematoxylin for 1 min, washed with tap water, and rehydrated in increasing ethanol concentrations from 50 to 100%, closed in a mounting medium with coverslips and analysed using an optical microscope (Axio Imager A1 Carl Zeiss).

The histological studies at the level of spinal cord compression revealed an axonal degeneration with partial or total loss of myelin (Fig.3A) as well as an increase in the number of microglia cells (Fig.3B), which later transformed into tissue macrophages responsible for phagocytosis and removal of debris from the damaged myelin sheaths. Macrophages loaded with phagocytized material (so-called “gitter cells”) were observed in the form of clusters of cells along blood vessels and around the “digestive chamber” (Fig.3B, Ds). Numerous “digestive chambers” (myelin sheath fragments being phagocytosed by macrophages) formed in the areas of the spinal cord that did not contain myelin, forming dysfunctional areas with a spongy structure typical for the wobbler syndrome (Fig.3E). The described changes generally affected the white matter- the lateral and dorsal funiculi, which are more sensitive to pressure or compression and are the first to undergo changes. In this horse, the axonal degeneration consisted of partial or complete loss of myelin sheaths or their transformation into swollen or spherical structures (Fig.3E). Moreover, the increase in perivascular collagen (Fig. 3B, C) was observed directly at the level of the spinal cord compression, whereas no gliosis (so-called astrocytic scar) was spotted, which may indicate an initial stage of the disease or its occurrence far from the place of compression. Immunohistochemical studies showed a negative transmembrane glycoprotein CD68 (−) monocyte response, by circulating and tissue macrophages such as microglia (Fig.3F) and a negative tumor necrosis factor alpha (TNF-α (−)) reaction (Fig.3G) – a.k.a. X.

Histological and immunohistochemical examination of equine spinal cord compression stained with hematoxylin and eosin (H&E) (a, d, e) and Masson’s trichrome staining (b, c): A- The difference between the white and grey matter at the site of spinal cord compression. b, C-The changes associated with an increased number of microglial cell infiltration and perivascular collagen D-Multiple digestive chambers forming a spongy structure with a macrophage infiltration (f) F- Expression of CD68 with few visible /several macrophages G- Expression of the tumor necrosis factor –alpha (TNF-α)


Wax on, melt off

Researchers from Drexel University, Purdue University and Oregon State University have discovered that adding paraffin oil to the mix for road concrete can give it the ability to melt ice and snow when temperatures fall. Credit: Drexel University

Drexel University researchers have made a discovery that could create roads that deice themselves during winter storms. Their secret?—Adding a little paraffin wax to the road's concrete mix.

In a paper recently published in journal Cement and Concrete Composites researchers, led by Yaghoob Farnam, PhD, an assistant professor in Drexel's College of Engineering, explain how substances like paraffin oil—known as "phase change materials" in chemistry—can be used in concrete to store energy and release it as heat when a road needs a melt-off.

Keeping roads open to travel is a persistent challenge during winter months, but efforts to make them safely passable—including the constant use of snow plows, deicing chemicals and road salt—tend to deteriorate the surface. The chemicals and road salts currently used to melt snow and ice can also have a deleterious environmental impact when surface runoff carries them into nearby ecosystems—which is pretty likely considering the state of Pennsylvania alone dumps more than 900,000 tons of it on roads each winter. So researchers have been searching for a better winter option than salting and plowing for some time.

Farnam's group in collaboration with researchers from Purdue University and Oregon State University, is among the first to demonstrate that using phase change materials as an environmentally friendly alternative can be just as effective as the standard salting and scraping methods.

"Phase change materials can be incorporated into concrete using porous lightweight aggregate or embedded pipes and when PCM transforms from liquid to solid during cooling events, it can release thermal heat that can be used to melt ice and snow," Farnam said. "By inhibiting the formation of ice and snow on the pavement or bridge surface, the use of PCM may reduce or eliminate the need for deicing chemicals/salts, snowplowing or both—thus saving money and positively influencing the environmental impact of such operations."

Paraffin oil, a common ingredient in candles, wax polishes, cosmetics and water-proofing compounds, was their material of choice for this endeavor because it is organic, widely available, chemically stable and relatively inexpensive. Like all phase change materials, it releases thermal energy when it changes its physical state, which means as temperatures drop and the oil begins to solidify it releases energy through latent heat of fusion. This means paraffin oil can be tailored to embed deicing capabilities in a road surface so that it becomes thermally active during snow events or when deicing is needed.

To test its snow and ice-melting ability, the team created a set of concrete slabs—one with paraffin-filled pipes inside, one containing porous lightweight aggregate that had been infused with paraffin, and a third reference slab without paraffin. Each was sealed in an insulated container and then covered with about five inches of lab-made "snow."

With temperatures inside the boxes held between 35-44 degrees Fahrenheit, both of the paraffin-treated slabs were able to completely melt the snow within the first 25 hours of testing, while the snow on the reference sample remained frozen. The slab with the paraffin-filled tubes melted the snow slightly faster than the one composed of paraffin-treated aggregate. Farnam suggests that this is because the paraffin inside the tubes is able to solidify more quickly—thus releasing its energy—because of the regular diameter of the pipes. While the diameter of the pores of the aggregate vary in size.

But in the group's second experiment, in which the ambient air temperature in the box was lowered to freezing before the snow was added, the paraffin-treated aggregate was more effective than the embedded pipes. This is because the capillary pore pressure delayed the freezing of the paraffin, thus allowing it to release its heat energy over a longer period of time.

"The gradual heat release due to the different pore sizes in porous light-weight aggregate is more beneficial in melting snow when concrete is exposed to variety of temperature changes when snow melting or deicing is needed," Farnam said. "We believe that using porous lightweight aggregate can be potential way of incorporating phase change materials in concrete as it is easy to be implemented in practice and can cover environmental conditions of various locations in the US dealing with snow, especially melting snow or deicing in roads and bridges in the Northeast."

One of the first uses of this infrastructure technology could be at airports, where keeping runways clear of snow and ice is vital and a perpetual challenge in the winter. The Federal Aviation Administration supported this research as part of its Heated Airport Pavements Project in its Partnership to Enhance General Aviation Safety, Accessibility and Sustainability program.