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Sıçan Ön ve Arka Ekstremite Kemiklerinin Prenatal ve Postnatal Dönemdeki Kemik Gelişimlerinin İkili Boyama Yöntemi ile Belirlenmesi

Year 2023, Volume: 7 Issue: 2, 190 - 199, 29.10.2023
https://doi.org/10.30565/medalanya.1262518

Abstract

Amaç: Çalışmamızda sıçan fetus ve yenidoğan iskeletinin ikili boyama yöntemi ile boyanarak, ön ve arka ekstremiteye ait kemiklerin morfolojik gelişimlerinin tespitini amaçladık.

Yöntem: Bu çalışmada 13 adet gebe sıçandan elde edilen fetüs ve yavrulardan 3'ü prenatal (16.,18. ve 20. gün) ve 4'ü postnatal (0, 3.,7. ve 12.gün) olmak üzere 7 grup oluşturuldu. Daha sonra ikili boyama yöntemiyle boyandı. Fetüs ve yavruların ön ve arka ekstremite görüntüleri stereo mikroskop altında incelenerek ossifikasyon bulguları belirlendi. ImageJ yazılımı kullanılarak toplam kemik ve ossifikasyon uzunlukları ile ossifikasyon alanları ölçüldü.

Bulgular: Fetüslerdeki ilk kıkırdak yıkımı clavicula, scapula, humerus, radius ve ulna’da gebeliğin 16. gününde; femur, tibia ve fibula’da ise gebeliğin 18. gününde görüldü. İlk kemikleşme merkezi clavicula, scapula ve humerusta gebeliğin 18. gününde; radius, ulna, femur, tibia, fibula ve 2-5 metatarsal kemiklerde gebeliğin 20. gününde görüldü. İkincil kemikleşme merkezi scapula ve humerusta 0. günde (doğum günü), ulna ve radiusta doğumdan sonra 7. günde, femur ve tibiada doğumdan sonra 12. günde görüldü. Çalışmada 20 günlük fetuslerde kemikleşme oranı humerus’ta %48.9, radius’ta %53.2, ulna’da %55.7, femur‘da %33.6, tibia’da %43.2, fibula’da %44.3’ken, doğumdan sonraki 12. günde humerus’ta %69.7, radius’ta %78.4, ulna’da %73.3, femur’da %63.5, tibia’da %75.5, fibula’da %69.2’e ulaştığı tespit edildi

Sonuç: Bu çalışmada, fetüslere ve yenidoğan yavrulara ait ön ve arka ekstremite kemiklerinin normal gelişim seyrindeki morfolojik değişimlerini ortaya koyduk. Bu sonuçların teratolojik çalışmalarda iskelet anomalilerinin (malformasyon, varyasyon ve diğer anomaliler) tespitine yönelik yapılacak çalışmalara ışık tutacağını ve yapılacak çalışmalardan elde edilecek bulguların daha kapsamlı değerlendirilmesine katkı sunacağını düşünmekteyiz.

Supporting Institution

Erciyes Üniversitesi Bilimsel Araştırma Projeleri Birimi

Project Number

TSY-11-3723

References

  • 1. De Sesso JM, Scialli AR. Bone development in laboratory mammals used in developmental toxicity studies. Birth Defects Res. 2018;110(15):1157–87. doi: 10.1002/bdr2.1350.
  • 2. De Schaepdrijver L, Delille P, Geys H, Boehringer-Shahidi C, Vanhove C. In vivo longitudinal micro-CT study of bent long limb bones in rat offspring. Reprod Toxicol. 2014;46:91–7. doi: 10.1016/j.reprotox.2014.03.004.
  • 3. Inouye M. Differential staining of cartilage and bone in fetal mouse skeleton by Alcian blue and Alizarin red S. Cong Anom. 1976;16:171–3.
  • 4. Chahoud I, Paumgartten FJR. Relationships between fetal body weight of Wistar rats at term and the extent of skeletal ossification. Brazilian J Med Biol Res. 2005;38(4):565–75. doi: 10.1590/S0100-879X2005000400010.
  • 5. Morini S, Continenza MA, Ricciardi G, Gaudio E, Pannarale L. Development of the Microcirculation of the Secondary Ossification Center in Rat Humeral Head. Anat Rec A Discov Mol Cell Evol Biol. 2004;278(1):419–27. doi: 10.1002/ar.a.20016.
  • 6. Yılmaz H, Ertekin T, Atay E, Nisari M, Susar Güler H, Al Ö, et al. Antioxidant role of melatonin against nicotine’s teratogenic effects on embryonic bone development. Iran J Basic Med Sci. 2018;21(8):787–93. doi: 10.22038/ijbms.2018.26705.6539.
  • 7. Yilmaz S, Göçmen AY, Üner AK, Akyüz E, Tokpinar A. The protective role of melatonin against the effect of caffeine on embryonic kidney. Turk Hij Deney Biyol Derg. 2020;77(1):51–8. doi: 10.5505/TurkHijyen.2020.77675.
  • 8. Abd El-Aziz GS, El-Fark MMO, Saleh HAM. The Prenatal Toxic Effect of Methylmercury on the Development of the Appendicular Skeleton of Rat Fetuses and the Protective Role of Vitamin E. Anat Rec (Hoboken). 2012;295(6):939–49. doi: 10.1002/ar.22485.
  • 9. Soysal H, Unur E, Düzler A, Karaca Ö, Ekinci N. Effects of intraperitoneal administration of the phenytoin on the skeletal system of rat fetus. Seizure. 2011;20(3):187–93. doi: 10.1016/j.seizure.2010.12.009.
  • 10. Hofmann T, Buesen R, Schneider S, van Ravenzwaay B. Postnatal fate of prenatal-induced fetal alterations in laboratory animals. Reprod Toxicol. 2016;61:177–85. doi: 10.1016/j.reprotox.2016.04.010.
  • 11. Aliverti V, Bonanomi L, Giavini E, Leone VG, Mariani L. The extent of fetal ossification as an index of delayed development in teratogenic studies on the rat. Teratology. 1979;20(2):237–42. doi: 10.1002/tera.1420200208.
  • 12. ImageJ n.d. https://imagej.nih.gov/ij/index.html (accessed September 7, 2023).
  • 13. Metwally A. Mansoor, Amal S.Sewelam, Maha M. Abdul Rahman Mohamad A. Sabry. A Model for the Study of Induced Skeletal Anomalies in Albino Rat Fetuses. J Am Sci. 2014;10(2):181–90.
  • 14. Nakajima M, Takahashi H, Nakazawa K, Usami M. Fetal cartilage malformation by intravenous administration of indium trichloride to pregnant rats. Reprod Toxicol. 2007;24(3-4):409–13. doi: 10.1016/j.reprotox.2007.06.001.
  • 15. Siddiqui MA, Janjua MZ. Effect of prenatal doxycycline administration on skeletal differentiation in long bones of albino rat. J Pak Med Assoc. 2002;52(5):211–4. PMID: 12174493
  • 16. Basal WT, Ahmed ART, Mahmoud AA, Omar AR. Lufenuron induces reproductive toxicity and genotoxic effects in pregnant albino rats and their fetuses. Sci Rep. 2020;10(1):19544. doi: 10.1038/s41598-020-76638-6.
  • 17. Burdan F, Baszczak-Szalak M, Róyo-Kalinowska I, Klepacz R, Dworzaski W, Róyo TK, et al. Early postnatal development of the lumbar vertebrae in male Wistar rats: Double staining and digital radiological studies. Folia Morphol (Warsz). 2016;75(1):1–13. doi: 10.5603/FM.a2015.0068.
  • 18. Bilir A, Atay E, Firat F, Kundakci YE. Investigation of developmental toxicity of favipiravir on fetal bone and embryonic development. Birth Defects Res. 2022;114(17):1092–100. doi: 10.1002/BDR2.2073.
  • 19. Atay E, Ertekin T, Yılmaz H, Güler HS, Al Ö, Nisari M, et al. Impact of prenatal exposure to bisphenol A on pregnant rats: Fetal bone development and immunohistochemistry implications. Toxicol Ind Health. 2019;35(2):119–35. doi: 10.1177/0748233718823146.
  • 20. Strong RM. The order, time, and rate of ossification of the albino rat (Mus norvegicus albinus) skeleton. Am J Anat. 1925;36(2):313–55. doi: 10.1002/aja.1000360206.
  • 21. Wright H V., Asling CW, Dougherty HL, Nelson MM, Evans HM. Prenatal development of the skeleton in long-evans rats. Anat Rec. 1958;130(4):673-89. doi: 10.1002/ar.1091300404. 
  • 22. Patton JT, Kaufman MH. The timing of ossification of the limb bones, and growth rates of various long bones of the fore and hind limbs of the prenatal and early postnatal laboratory mouse. J Anat. 1995 Feb;186 ( Pt 1)(Pt 1):175-85. PMID: 7649813
  • 23. Maximow AA, Bloom W, Fawcett DW. A Textbook of Histology. 8th ed. Saunders; 1965. New York.
  • 24. Campion SN, Nowland WS, Gropp K, Liu CN, Ritenour HN, Syed J, et al. Assessment of postnatal femur development in Wistar Han rats. Birth Defects Res. 2022;114(15):863–72. doi: 10.1002/BDR2.2071.
  • 25. Hedberg A, Messner K, Persliden J, Hildebrand C. Transient local presence of nerve fibers at onset of secondary ossification in the rat knee joint. Anat Embryol (Berl). 1995;192(3):247–55. doi: 10.1007/BF00184749.
  • 26. Saeidinezhad M, Razban V, Safizadeh H, Ezzatabadipour M. Effects of maternal consumption of morphine on rat skeletal system development. BMC Musculoskelet Disord. 2021;22(1):435. doi: 10.1186/s12891-021-04321-6.
  • 27. Carney EW, Kimmel CA. Interpretation of skeletal variations for human risk assessment: Delayed ossification and wavy ribs. Birth Defects Res B Dev Reprod Toxicol. 2007;80(6):473–96. doi: 10.1002/bdrb.20133.
  • 28. Hayasaka I, Tamaki F, Uchıyama K, Kato Z, Murakami K. Azosemide‐Induced Fetal Wavy Ribs and Their Disappearance after Birth in Rats. Congenit Anom (Kyoto). 1985;25(2):121–7. doi: 10.1111/j.1741-4520.1985.tb01003.x.

Determination of Bone Developments of Rat Anterior and Posterior Extremity Bones in Prenatal and Postnatal Period by Double Staining Method

Year 2023, Volume: 7 Issue: 2, 190 - 199, 29.10.2023
https://doi.org/10.30565/medalanya.1262518

Abstract

Aim: In our study, we aimed to determine the morphological development of the bones of the anterior and posterior extremity by staining the rat fetus and offspring skeletons with the double staining method.

Method: In the current study, seven groups three prenatal (16th, 18th, and 20th days) and four postnatal (0th, 3th, 7th and 12th days) were formed from the foetuses and offsprings obtained from 13 pregnant rats. Then, it was stained with double staining method. Anterior and posterior extremity images of the fetuses and offsprings were examined under a stereo microscope, and ossification findings were determined. Total bone and ossification lengths as well as ossification areas were measured using the ImageJ software.

Results: The first cartilage destruction in fetuses occurred on the 16th day of pregnancy in the clavicle, scapula, humerus, radius and ulna; It was seen in the femur, tibia and fibula on the 18th day of pregnancy. The first ossification centres were in the clavicle, scapula and humerus on the 18th day of pregnancy; It was seen in the radius, ulna, femur, tibia, fibula and 2-5 metatarsal bones on the 20th day of pregnancy. The secondary ossification centre was seen on the 0th day (birthday) in the scapula and humerus, on the 7th day after birth in the ulna and radius, and on the 12th day after birth in the femur and tibia. In the study, while the ossification rate in 20-day-old foetuses was 48.9% in the humerus, 53.2% in the radius, 55.7% in the ulna, 33.6% in the femur, 43.2% in the tibia, 44.3% in the fibula, it was determined that it reached 69.7% in the humerus, 78.4% in the radius, 73.3% in the ulna, 63.5% in the femur, 75.5% in the tibia, and 69.2% in the fibula on the 12th day after birth.

Conclusion: In this study, we revealed the morphological changes of the anterior and posterior extremity bones of fetuses and offsprings in the normal developmental course. We think that these results will shed light on the studies to be conducted on the detection of skeletal anomalies in teratological studies and contribute to a more comprehensive evaluation of the findings to be obtained from the studies to be conducted.

Project Number

TSY-11-3723

References

  • 1. De Sesso JM, Scialli AR. Bone development in laboratory mammals used in developmental toxicity studies. Birth Defects Res. 2018;110(15):1157–87. doi: 10.1002/bdr2.1350.
  • 2. De Schaepdrijver L, Delille P, Geys H, Boehringer-Shahidi C, Vanhove C. In vivo longitudinal micro-CT study of bent long limb bones in rat offspring. Reprod Toxicol. 2014;46:91–7. doi: 10.1016/j.reprotox.2014.03.004.
  • 3. Inouye M. Differential staining of cartilage and bone in fetal mouse skeleton by Alcian blue and Alizarin red S. Cong Anom. 1976;16:171–3.
  • 4. Chahoud I, Paumgartten FJR. Relationships between fetal body weight of Wistar rats at term and the extent of skeletal ossification. Brazilian J Med Biol Res. 2005;38(4):565–75. doi: 10.1590/S0100-879X2005000400010.
  • 5. Morini S, Continenza MA, Ricciardi G, Gaudio E, Pannarale L. Development of the Microcirculation of the Secondary Ossification Center in Rat Humeral Head. Anat Rec A Discov Mol Cell Evol Biol. 2004;278(1):419–27. doi: 10.1002/ar.a.20016.
  • 6. Yılmaz H, Ertekin T, Atay E, Nisari M, Susar Güler H, Al Ö, et al. Antioxidant role of melatonin against nicotine’s teratogenic effects on embryonic bone development. Iran J Basic Med Sci. 2018;21(8):787–93. doi: 10.22038/ijbms.2018.26705.6539.
  • 7. Yilmaz S, Göçmen AY, Üner AK, Akyüz E, Tokpinar A. The protective role of melatonin against the effect of caffeine on embryonic kidney. Turk Hij Deney Biyol Derg. 2020;77(1):51–8. doi: 10.5505/TurkHijyen.2020.77675.
  • 8. Abd El-Aziz GS, El-Fark MMO, Saleh HAM. The Prenatal Toxic Effect of Methylmercury on the Development of the Appendicular Skeleton of Rat Fetuses and the Protective Role of Vitamin E. Anat Rec (Hoboken). 2012;295(6):939–49. doi: 10.1002/ar.22485.
  • 9. Soysal H, Unur E, Düzler A, Karaca Ö, Ekinci N. Effects of intraperitoneal administration of the phenytoin on the skeletal system of rat fetus. Seizure. 2011;20(3):187–93. doi: 10.1016/j.seizure.2010.12.009.
  • 10. Hofmann T, Buesen R, Schneider S, van Ravenzwaay B. Postnatal fate of prenatal-induced fetal alterations in laboratory animals. Reprod Toxicol. 2016;61:177–85. doi: 10.1016/j.reprotox.2016.04.010.
  • 11. Aliverti V, Bonanomi L, Giavini E, Leone VG, Mariani L. The extent of fetal ossification as an index of delayed development in teratogenic studies on the rat. Teratology. 1979;20(2):237–42. doi: 10.1002/tera.1420200208.
  • 12. ImageJ n.d. https://imagej.nih.gov/ij/index.html (accessed September 7, 2023).
  • 13. Metwally A. Mansoor, Amal S.Sewelam, Maha M. Abdul Rahman Mohamad A. Sabry. A Model for the Study of Induced Skeletal Anomalies in Albino Rat Fetuses. J Am Sci. 2014;10(2):181–90.
  • 14. Nakajima M, Takahashi H, Nakazawa K, Usami M. Fetal cartilage malformation by intravenous administration of indium trichloride to pregnant rats. Reprod Toxicol. 2007;24(3-4):409–13. doi: 10.1016/j.reprotox.2007.06.001.
  • 15. Siddiqui MA, Janjua MZ. Effect of prenatal doxycycline administration on skeletal differentiation in long bones of albino rat. J Pak Med Assoc. 2002;52(5):211–4. PMID: 12174493
  • 16. Basal WT, Ahmed ART, Mahmoud AA, Omar AR. Lufenuron induces reproductive toxicity and genotoxic effects in pregnant albino rats and their fetuses. Sci Rep. 2020;10(1):19544. doi: 10.1038/s41598-020-76638-6.
  • 17. Burdan F, Baszczak-Szalak M, Róyo-Kalinowska I, Klepacz R, Dworzaski W, Róyo TK, et al. Early postnatal development of the lumbar vertebrae in male Wistar rats: Double staining and digital radiological studies. Folia Morphol (Warsz). 2016;75(1):1–13. doi: 10.5603/FM.a2015.0068.
  • 18. Bilir A, Atay E, Firat F, Kundakci YE. Investigation of developmental toxicity of favipiravir on fetal bone and embryonic development. Birth Defects Res. 2022;114(17):1092–100. doi: 10.1002/BDR2.2073.
  • 19. Atay E, Ertekin T, Yılmaz H, Güler HS, Al Ö, Nisari M, et al. Impact of prenatal exposure to bisphenol A on pregnant rats: Fetal bone development and immunohistochemistry implications. Toxicol Ind Health. 2019;35(2):119–35. doi: 10.1177/0748233718823146.
  • 20. Strong RM. The order, time, and rate of ossification of the albino rat (Mus norvegicus albinus) skeleton. Am J Anat. 1925;36(2):313–55. doi: 10.1002/aja.1000360206.
  • 21. Wright H V., Asling CW, Dougherty HL, Nelson MM, Evans HM. Prenatal development of the skeleton in long-evans rats. Anat Rec. 1958;130(4):673-89. doi: 10.1002/ar.1091300404. 
  • 22. Patton JT, Kaufman MH. The timing of ossification of the limb bones, and growth rates of various long bones of the fore and hind limbs of the prenatal and early postnatal laboratory mouse. J Anat. 1995 Feb;186 ( Pt 1)(Pt 1):175-85. PMID: 7649813
  • 23. Maximow AA, Bloom W, Fawcett DW. A Textbook of Histology. 8th ed. Saunders; 1965. New York.
  • 24. Campion SN, Nowland WS, Gropp K, Liu CN, Ritenour HN, Syed J, et al. Assessment of postnatal femur development in Wistar Han rats. Birth Defects Res. 2022;114(15):863–72. doi: 10.1002/BDR2.2071.
  • 25. Hedberg A, Messner K, Persliden J, Hildebrand C. Transient local presence of nerve fibers at onset of secondary ossification in the rat knee joint. Anat Embryol (Berl). 1995;192(3):247–55. doi: 10.1007/BF00184749.
  • 26. Saeidinezhad M, Razban V, Safizadeh H, Ezzatabadipour M. Effects of maternal consumption of morphine on rat skeletal system development. BMC Musculoskelet Disord. 2021;22(1):435. doi: 10.1186/s12891-021-04321-6.
  • 27. Carney EW, Kimmel CA. Interpretation of skeletal variations for human risk assessment: Delayed ossification and wavy ribs. Birth Defects Res B Dev Reprod Toxicol. 2007;80(6):473–96. doi: 10.1002/bdrb.20133.
  • 28. Hayasaka I, Tamaki F, Uchıyama K, Kato Z, Murakami K. Azosemide‐Induced Fetal Wavy Ribs and Their Disappearance after Birth in Rats. Congenit Anom (Kyoto). 1985;25(2):121–7. doi: 10.1111/j.1741-4520.1985.tb01003.x.
There are 28 citations in total.

Details

Primary Language English
Subjects Clinical Sciences (Other)
Journal Section Research Article
Authors

Mustafa Öztürk 0000-0002-7797-1353

Erdoğan Unur 0000-0003-2033-4350

Niyazi Acer 0000-0002-4155-7759

Tolga Ertekin 0000-0003-1756-4366

Serife Alpa 0000-0001-8665-3632

Mesut Meker 0000-0002-2275-9814

Yahya Tahta 0000-0001-7513-5872

Project Number TSY-11-3723
Early Pub Date October 20, 2023
Publication Date October 29, 2023
Submission Date March 9, 2023
Acceptance Date September 28, 2023
Published in Issue Year 2023 Volume: 7 Issue: 2

Cite

Vancouver Öztürk M, Unur E, Acer N, Ertekin T, Alpa S, Meker M, Tahta Y. Determination of Bone Developments of Rat Anterior and Posterior Extremity Bones in Prenatal and Postnatal Period by Double Staining Method. Acta Med. Alanya. 2023;7(2):190-9.

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