Transcardial perfusion versus immersion fixation for assessment of peripheral nerve regeneration

Kasukurthi, Rahul; Brenner, Michael; Moore, Amy M.; Moradzadeh, Arash; Ray, Wilson Z.; Santosa, Katherine B.; Mackinnon, Susan E.; Hunter, Daniel A.

doi:10.1016/j.jneumeth.2009.08.019

Cited by 40 publications

(35 citation statements)

References 32 publications

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“…While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate 5,7 . Often, changes in response to hypoxia begin before the tissue can be preserved…”

Section: Introductionmentioning

confidence: 99%

Whole Animal Perfusion Fixation for Rodents

Gage

Kipke

Shain

2012

JoVE

419

315

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The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate 5,7 . Often, changes in response to hypoxia begin before the tissue can be preserved 12. The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures 3 . It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively. Video LinkThe video component of this article can be found at https://www.jove.com/video/3564/ Protocol

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Section: Introductionmentioning

confidence: 99%

Whole Animal Perfusion Fixation for Rodents

Gage

Kipke

Shain

2012

JoVE

419

315

View full text Add to dashboard Cite

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“…A proper fixation protocol is crucial, as well for the studies on histomorphometric evaluation of peripheral nerve fibers particularly concerning the quantitative features such as axon number, axonal cross-sectional area and 2,4 . Most commonly used fixation protocols in these studies are immersion and perfusion fixation techniques 5,14,16 . Although transcardial perfusion has been approved as the most effective method it indeed has several disadvantages.…”

Section: Discussionmentioning

confidence: 99%

“…It occupies plenty of time, labor and definitely should be performed by expertised staff. Biosafety of the procedure is subject to interrogation, as well, since large amount of fixatives including suspected carcinogens are applied 5,17 . A shortfall of transcardial perfusion is that the peripheral nerve can be curved by muscular contractions during the procedure and may thus be fixed in a curved shape, which makes sectioning tricky 1 .…”

Section: Discussionmentioning

confidence: 99%

“…Accurate analysis, however, depends on high-quality fixation of peripheral nerve tissue. The objective of fixation is to preserve the tissue sample as close to its natural form as possible, though even subtle changes in the intrinsic structure of the tissue sample, inevitably give rise to artifacts 5 .…”

Section: Introductionmentioning

confidence: 99%

“…Transcardial perfusion and immersion are the two most common methods used for fixation, the former being accepted as the gold standard for the majority of the studies of neural tissue although some researchers pointed out the nonessentiality of transcardial perfusion, which was considered to be replaced by immersion of nerve specimens in the fixative agent after being dissected 5 .…”

Section: Introductionmentioning

confidence: 99%

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Comparison of Fixation Methods for Peripheral Nerve Fiber

Ulkay¹,

Aktaş²,

Aksoy³

et al. 2013

Kafkas Univ Vet Fak Derg

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Accurate fixation is a must for the assessment of myelinated nerve fiber morphology and transcardial perfusion and immersion methods are the most commonly used fixation techniques. In the present study we designed a new fixation technique for the histomorphometric and stereological evaluation of sciatic nerve fiber and referred it as instillation fixation. The method involved preliminary in situ fixation of the nerve sample without dissecting it from the animal body followed by a complete conventional fixation protocol. The objective of this study was to compare the three fixation techniques with each other for fixation artifacts. Eighteen female Wistar albino rats constituted the study material. The animals were allocated into three experimental groups corresponding to different fixation methods (immersion, instillation and transcardial perfusion, respectively). Quantitative assessments of nerve samples harvested from the animals of each group included the number of total myelinated axons, normal myelinated axons, alterations in myelin compaction, myelinated axons with irregular fiber shape, and with myelin loops and g ratio. Results revealed that normal myelinated axons were markedly lower in the immersion fixation group compared to those of instillation and transcardial perfusion. Moreover a significant decrease was noted with respect to alterations in myelin compaction in the instillation fixation group. In contrast, no significant difference was observed in myelin thickness and axon cross sectional area. In conclusion instillation fixation technique proved to be a valid and simple method for the assessment of peripheral nerve morphology for further analyses in a rat model. Keywords: Rat, Sciatic nerve, Fixation method Periferal Sinir Tespit Yöntemlerinin KarşılaştırılmasıÖzet Doğru bir tespit işlemi miyelinli periferik sinirlerin morfolojik değerlendirmesi için ön koşuldur. Kalp perfüzyonu ve daldırma en yaygın kullanılan tespit yöntemlerindendir. Çalışmamızda bu metotlara ek olarak damlatma tekniği olarak adlandırılan yeni bir tespit metodu uygulandı. Sinir doku örneği canlı hayvan üzerinden çıkarılmadan ön tespit işlemine tabi tutulduktan sonra rutin tespit işlemleri tamamlandı ve üç tespit yönteminin tespit artefaktları açısından birbirleriyle histomorfometrik ve stereolojik olarak karşılaştırılması gerçekleştirildi. On sekiz adet dişi Wistar albino sıçan kullanıldı. Hayvanlar sırasıyla daldırma, damlatma ve kalp perfüzyonu olmak üzere üç eşit gruba ayrıldı. Her bir gruptaki hayvanlara ait siyatik sinir örneklerinin kantitatif değerlendirilmesi toplam miyelinli akson sayısı, normal miyelinli akson sayısı, miyelin kompaksiyonundaki değişimler içeren aksonların sayısı, düzensiz demet yapısı ile kıvrımlı miyelin halkaları içeren miyelinli aksonların sayısı ve g oranları hesaplanarak gerçekleştirildi. Elde edilen bulgulara göre daldırma tespit grubundaki normal miyelinli akson sayısında, damlatma ve kalp perfüzyonu grupları ile karşılaştırıldığında belirgin bir düşüş belirlendi. Bununla beraber damlatma tesp...

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Anatomy and Processing of Peripheral Nerve Tissues

Valentine

Bolon

2011

Fundamental Neuropathology for Pathologists and Toxicologists

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Transcardial perfusion versus immersion fixation for assessment of peripheral nerve regeneration

Cited by 40 publications

References 32 publications

Whole Animal Perfusion Fixation for Rodents

Whole Animal Perfusion Fixation for Rodents

Comparison of Fixation Methods for Peripheral Nerve Fiber

Anatomy and Processing of Peripheral Nerve Tissues

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