A Morphological and Stereological Study on Cervical Spinal Cord of One and Five Months Age Male Rat

In this study volume density of gray and white matter of cervical segments of spinal cords of rats were investigated using stereological method. Twelve male Wistar albino rats were used in the study as two different age groups 1 month and 5 months. All animals were fixed by perfusing 10% buffered formalin. Rats were dissected and spinal cords of rats were removed. In the study cervical segments of 1 and 5 months age rats were obtained. One of the first 15 section were selected randomly when the sections were taken. And following every 50th section was determined by systematic random sampling. Thus, 8-10 sections of 5µm thickness were obtained from cervical segments of each animal’s medulla spinalis. These sections were stained by hemotoxylin eosin and they were photographed at microscope. Densities of volumes of all tissue of cervical segments of whole spinal cord and white and gray matters were calculated using dotted area ruler by Cavalieri Principle. SHTEREOM 1.5 package programme was used for counting dotted area. In addition, the volume vales of total cervical segment, volume values of the white matter and the gray matter and the ratios of these volume values to each other were evaluated in the study.

Anahtar Kelimeler:

Cervical segment, rat, spinal cord, stereology, volume

P1 transduction method to construct Escherichia coli mutant strains lacking more than one gene

Assigning gene functions and performing phenotypic studies of the bacteria require the construction of specific mutants that at times should lack more than one gene due to the redundancy of particular gene activities. P1 phage is a generalized transducing phage that can be used in constructing double, triple or multiple mutants of Escherichia coli. This phage is able to mistakenly package random fragments of E. coli chromosome (~2 % of the genome into each particle) into the phage head during phage development. The phages with E. coli DNA can inject that DNA into recipient cells. Given homology between donor and recipient, and a selectable marker, transductants can be obtained. For transduction, a virulent derivative of phage P1 is preferably used because this form cannot integrate into the bacterial genome. The virulent phage can only develop using the lytic mode of development and produces phage progeny upon lysis of the host cell. This technique is based on recombination technology, allowing the DNA fragments of the E. coli genome to be transported among the strains. In this study, we optimized the P1 transduction methodology, which we aim to present to the Turkish scientific community in the native language with the experimental flow and sufficient details. For this reason, a number of four single gene mutants obtained from the KEIO collection were used. First, kanamycine (Kan) gene cassettes were removed from each mutant strain to create a recipient strain that is kan sensitive. The other four KEIO mutant strains with KanR cassettes were used as the donor strains and P1 phage were cultured with these strains and the lysates were obtained. These lysates and recipient strains were then transduced to integrate the DNA fragments of the lysates into the recipient strain genome. Upon selection with kanamycine, new mutant strains, double mutants are obtained. As a result, four double mutants namely DtsgAugpA, DyhdTompA, DugpAmdtG and DaroGmalF were obtained and the details pertaining to the protocols and results are presented and discussed.

Keywords:

P1 phage, transduction, Escherichia coli, double mutants,

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[1]. Ide T. 2003. Animal Models. Laboratory Animals, Basic Principles of Science. Turkish translation, Zutphen LFM, Baumans V, Beynen AC. In: Ide T, Translation ed. Medipres Publications, Ozkan Typography, Ankara.
[2]. Deniz G., Oral B. 2011. Experimental Animal Modeling Workshop. The Official Turkish Journal of Society of Immunology. 1, 10-18.
[3]. Dursun N. 2000. Veterinary Anatomy III. Medisan Publishing, 15-23pp. Ankara.
[4]. Arinci K., Elhan A. 2001. Anatomy. Vol 1. 3rd ed., Gunes Publishing, 59-65p. Ankara.
[5]. Zeman W., Maitland J.M. 1963. Cragie's Neuroanatomy of the Rat (Revised and expanded). In Academic Press Inc, 230p. New York.
[6]. Dursun N. 2008. Veterinary Anatomy I. 7th ed., Medisan Publishing, 15-67 pp. Ankara.
[7]. Bahadir A., Yildiz H. 2010. Veterinary anatomy movement system and internal organs. 3rd ed., Ezgi Publishing, 37-56p. Istanbul.
[8]. Unur E., Ulger H., Ekinci N. 2002. Anatomy. Ufuk Publishing, Kayseri.
[9]. Schoenen J., Faull R.L.M. 2004. Spinal cord cyto and chemoarchitecture. The human nervous system.. In: George Paxinos JKM, 2nd ed., Elsevier Academic Press, 190-228 pp. London.
[10]. Tanyolac A. 1993. Special Histology. 23-24p.
[11]. Cruz-Orive L.M., Weibel E.R. 1990. Recent stereological methods for cell biology: a brief survey. Lung Cellular and Molecular Physiology. American Journal of Psychology, 258, 148-156.
[12]. Baddeley A.J. 1991. Stereology. Spatial statistics and digital image analysis. In: DC: Natural Research Company Ed., Washington.
[13]. Gundersen H.J. 1986. Stereology of arbitrary particles. A review of unbiased number and size estimators and the presentation of some new ones. In: memory of William R. Thompson, Journal of Microscopy, 143, 3-45.
[14]. Gundersen H.J, Jensen E.B. 1987. The efficiency of systematic sampling in stereology and its prediction. Journal of Microscopy, 147, 229-263.
[15]. Howard C.V., Reed M.G. 1998. Unbiased Stereology: Three-dimensional measurement in microscopy. 1st ed., BIOS Scientific Publishers, UK.
[16]. Garcia-Finana M., Cruz-Orive L.M., Mackay C.E. 2003. Comparison of MR imaging against physical sectioning to estimate the volume of human cerebral compartments. Neuroimage. 18, 505-516.
[17]. Turgut M., Turkkanitunc A., Aslan H. 2007. Effect of pinealectomy on the morphology of the chick cervical spinal cord: A stereological and histopathological study. Brain Research, 1129, 166-173.
[18]. Slomianka L., West M.J. 2005. Estimators of the precision of stereological estimates: An example based on the CA1 pyramidal cell layer of rats. Neuroscience, 136, 757-767.
[19]. Gundersen H.J.G., Jensen E.B.V., Kıeu K. 1999. The efficiency of systematic sampling in stereology- reconsidered. Journal of Microscopy, 193, 199-211.
[20]. Canan S., Sahin B., Odacı E. 2002. A stereological method used to calculate total volume, volume density and volume ratios: Cavalieri principle. Clincal Turkey Journal of Medical Science, 22, 7-14.
[21]. Gundersen H.J.G., Bendtsen T.F., Korbo L. 1988. Some new, simple, and efficient stereological methods and their use in pathological research and diagnosis. Acta Pathologica Et Microbiologica Scandinavica Section A-Pathology, 96, 379-394.
[22]. Royet J.P. 1991. Stereology: a method for analysing images. Prog Neurobiology. 37, 433-474.
[23]. Sahin B., Aslan H., Unal B. (2001) Brain volumes of the lamb rat and bird do not show hemispheric asymmetry: a stereological study. Image Analysis and Stereology, 20, 9-13.
[24]. Mis L., Eski F., Yeltekin A.C. 2018. Plasma Macro and Trace Element Levels of Male Rats Vaccinated with GnRH Hormone. Fresenismus Environmental Bulletin, 27, 5085-5090.
[25]. Bancroft J.D., Stivens A., Turner D.R. 1996. Theory and practice of histological technique. 4th ed., 15, 76-81 p. Churchill Livingstone.
[26]. Odaci E., Bahadir A., Yildirim S. 2005. Volume Calculation and Clinical Use of Computerized Tomography and Magnetic Resonance Images Using Cavalieri Principle. Turkish Journal of Veterinary and Animal Science, 25, 421-428.
[27]. Begum F., Zhu W., Namaka M.P. 2010. A novel decalcification method for adult rodent bone for histological analysis of peripheral-central nervous system connections. Journal of Neuroscience Methods, 187, 59-66.
[28]. Waibl H. 1973. Zur Topographie der Medulla spinalis der Albinoratte (Rattus norvegicus). Advances in Anatomy, Embryology and Cell Biology, 47, 5-42.
[29]. Fritzgerald M.J.T. 1987. Anatomy and embriyology of the laboratory rat. Journal of Anatomy. 153, 256.
[30]. Paxinos G. 2004. The Rat Nervous System. 3rd ed. Prince of Wales Medical Research Instıtute The University of New South Wales Sydney, Australia.
[31]. Bolat D., Tipirdamaz S. 2011. Examination of spinal cord by stereological methods in leghorn chickens. University of Selcuk, Health Sciences Institute, Doctorate Thesis, Konya.
[32]. Baumel E. 1966. Comparable neurology of domestic animals. Turkish Journal of Veterinary and Animal Science, 203-289.
[33]. Haziroglu R.M., Orhan I.O., Yildiz D. 2001. Morphology of the spinal cord in the chicken, duck and pigeon. Turkish Journal of Veterinary and Animal Science, 25, 913-920.
[34]. Nickel R., Schummer A., Seiferle E. 1977. Anatomy of the domestic birds. Parey New york: Springer-Verlag, Berlin.
[35]. Rahmanifar F., Mansouri S., Ghazi S. 2008. Histomorphometric study of the spinal cord segments in the chick and adult male ostrich (Struthio camelus). Iranian Journal of Veterinary Research, 4, 336-340.
[36]. Braun A. 1950. Der segmentale feinbau des rückenmarks des pferdes. Acta Anatomica Basel. 10, 1-76.
[37]. Thomas C., Combs C.M. 1965. Spinal cord segments. B. Gross structure in the adult monkey. American Journal of Anatomy, 116, 205-216.
[38]. Ocal M., Haziroglu R.M. 1988. Comparative morphological studies on the spinal cord of the donkey (Equus asinus L.). Examination of transversal sections of segments. Veterinary Journal of Ankara University. 35, 55-68.
[39]. Ko H., Park J.H., Shin Y.B. 2004. Gross quantitayive measurements of spinal cord in human. Spinal Cord, 42, 35-40.
[40]. Portiansky L., Barbeito C.G., Goya R.G. 2004. Morphometry of cervical segments grey matter in the male rat spinal cord. Journal of Neuroscience Methods, 139, 217-229.