लेजर प्रवृत्त Zebrafish भ्रूण कंकाल की मांसपेशी की चोट

Summary

यहाँ प्रस्तुत विधि उच्च ऊर्जा लेजर दालों और ये समय के साथ और उनके वसूली चोटों के बाद के विश्लेषण के साथ रहते zebrafish भ्रूण की सटीक चोट शामिल हैं. हम यह भी पता चलता है कि आनुवंशिक एकल लेबल या कंकाल की मांसपेशी कोशिकाओं के समूहों के दौरान और लेजर प्रकाश प्रेरित क्षति के बाद लगाया जा सकता है.

Abstract

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see ¹). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel² or sapje³) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes⁴. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy⁵. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally¹. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo⁶, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells⁷.

Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.

Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail^{8, 9}. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish^{10, 11}.

Protocol

1. एकल कक्षों लेबलिंग Β-actin एक प्रमोटर के नियंत्रण के तहत प्लाज्मिड एन्कोडिंग या किसी भी GFP-संलयन प्रोटीन GFP के साथ एक कोशिका चरण भ्रूण इंजेक्षन. विकास के दौरान, GFP तो एक मोज़ेक फैशन में व्यक्त की है….

Representative Results

लेजर की मध्यस्थता चोट immobilized 1 दिन पुराने भ्रूण पर प्रदर्शन किया था. चित्र 1 में दिखाया गया है, कुछ लेजर दालों एक छोटा सा घाव उत्पन्न, आसानी से क्षतिग्रस्त, coiled, Actin अमीर myofibrils कि आम तौर पर कायखंड सीमाओं के ?…

Discussion

लेजर की मध्यस्थता की चोट कोशिकाओं ablating क्रम में zebrafish भ्रूण में नियंत्रित परिस्थितियों में उत्थान अध्ययन के द्वारा एक वांछित आकार के घाव inflicting के लिए एक शक्तिशाली तरीका है. विशेष रूप से, कोशिकाओं ठीक लक्षि?…

Materials

Heating block
Pair #5 forceps	Dumont
Glass slides	Menzel		76 x 26 mm
Coverslips	Roth		50 x 24 mm #1
Petroleum jelly
Stereomicroscope	Leica		MZFLIII
Micropoint laser	Andor Technology
Fluorescence microscope	Zeiss		Axioplan II
Metamorph software	Molecular devices
			Reagents Low-melting point agarose ( #50081, Lonza) Tricaine stock solution: 400 mg Tricaine (#A-5040, Sigma-Aldrich ) / 100 ml dH2O pH 9.0 E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4)