Summary

البصري مستدعى تسجيل المحتملين في نموذج الفئران التجريبية العصب البصري إزالة الميالين

Published: July 29, 2015
doi:

Summary

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

Abstract

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Introduction

Optic neuritis is one of the most common form of optic neuropathy, causing complete or partial loss of vision1. Histologically, it is featured by inflammatory demyelination, retinal ganglion cell axonal loss and varying degrees of remyelination in the optic nerve2. Optic neuritis is usually the manifest onset of multiple sclerosis. The visual evoked potential (VEP) is a non-invasive tool for investigating the function of the visual system. It reflects the post-retinal function from the retina to the primary visual cortex and is affected in many optic nerve disease conditions3. The VEP has been predominantly used in optic neuritis patients to assess the integrity of the visual pathway4.

The latency of VEP, which reflects the velocity of signal conduction along the visual pathway, is considered to be an accurate measurement of the level of myelin associated changes in the optic nerve5; while the amplitude of VEP is believed to be closely correlated with axonal damage of the retinal ganglion cells (RGC)6. This hypothesis has been fairly well established using the rat model of lysolecithin-induced optic nerve demyelination5.

Here, we explicate a comprehensive protocol of optic nerve microinjection technique in rodents, which can minimise the surgical manipulation-related damage to the nerve per se as well as to the adjacent tissues such as extraocular muscles and blood vessels. Also, the skull electrode implantation surgery has been described for VEP recording in animals7. The VEP recordings can be repeatedly carried out on animals over a period of time to assess demyelination/remyelination related changes as well as impact on axonal integrity in the optic nerve.

Protocol

وأجريت جميع الإجراءات التي تنطوي على الحيوانات وفقا للقانون الاسترالي من الممارسة لرعاية واستخدام الحيوانات لأغراض علمية والمبادئ التوجيهية لبيان ARVO لاستخدام الحيوانات في العيون والبحوث الرؤية، وتمت الموافقة من قبل: بيان الأخلاق لجنة أخلاقيات الحيوان من جامعة ما…

Representative Results

وتظهر آثار استنساخه VEP داخل الدورات في الشكل 1 و تأخير كبير في الكمون N1 يمكن أن ينظر بعد حقن العصب البصري. ويمكن ملاحظة آفات العصب البصري جزئية من إزالة الميالين على أقسام النسيجية باستخدام Luxol تلطيخ الأزرق سريع 5 الشكل 2 يوضح مقطع تمثيلي مع آفة ?…

Discussion

The optic nerve is very susceptible to mechanical damage. Optic nerve crush injury over a duration of 1 s can lead to about 75% loss of RGC over a period of 2 weeks10. Therefore, extreme care is required while performing the surgical procedures. According to the authors’ experience, it is much better to adapt a blunt dissection approach to expose and make way through the tissues around the optic nerve along the orientation of the nerve, rather than penetrating in a perpendicular orientation to the optic …

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

وأيد هذه الدراسة من قبل معهد بحوث أمراض العيون أستراليا (ORIA). نشكر الأستاذ Algis Vingrys والدكتور بانغ بوي، جامعة ملبورن، لمساعدتنا في البداية لتطوير تقنية تسجيل VEP.

Materials

Ketamine 100 mg/ml (Ketamil) Troy Laboratories AC 116
Medetomidine 1 mg/ml (Domitor) Pfizer sc-204073
Tropicamide 1.0% (Mydriacyl) Alcon sc-202371
Homoeothermic blanket system Harvard Apparatus NC9203819
Impedance meter  Grass F-EZM5
Screw electrodes  Micro Fasteners M1.0×3mm Csk Slot M/T 304 S/S
Subdermal needle electrodes  Grass F-E3M-72
Rapid Repair  DeguDent GmbH
Light-emitting diode  Nichia NSPG300A
Bioamplifier CWE, Inc. BMA-400
CED system Cambridge Electronic Design, Ltd. Power1401
Hamilton syringe  Hamilton 87930
Lysolecithin Sigma L4129
Evan’s blue  Sigma E2129

Referencias

  1. Balcer, L. J. Clinical practice. Optic neuritis. N Engl J Med. 354 (12), 1273-1280 (2006).
  2. Lassmann, H., Waxman, S. G. . Multiple sclerosis as a neuronal disease. , 153-164 (2005).
  3. Fahle, M., Bach, M., Heckenlively, J., Arden, G. . Principles and practice of clinical electrophysiology of vision. , 207-234 (2006).
  4. Halliday, A. M., McDonald, W. I., Mushin, J. Delayed visual evoked response in optic neuritis. Lancet. 1, 982-985 (1972).
  5. You, Y., Klistorner, A., Thie, J., Graham, S. L. Latency delay of visual evoked potential is a real measurement of demyelination in a rat model of optic neuritis. Invest Ophthalmol Vis Sci. 52 (9), 6911-6918 (2011).
  6. You, Y., Klistorner, A., Thie, J., Gupta, V. K., Graham, S. L. Axonal loss in a rat model of optic neuritis is closely correlated with visual evoked potential amplitudes using electroencephalogram based scaling. Invest Ophthalmol Vis Sci. 53, 3662 (2012).
  7. You, Y., Klistorner, A., Thie, J., Graham, S. L. Improving reproducibility of VEP recording in rats: electrodes, stimulus source and peak analysis. Doc Ophthalmol. 123 (2), 109-119 (2011).
  8. Heiduschka, P., Schraermeyer, U. Comparison of visual function in pigmented and albino rats by electroretinography and visual evoked potentials. Graefes Arch Clin Exp Ophthalmol. 246 (11), 1559-1573 (2008).
  9. You, Y., Thie, J., Klistorner, A., Gupta, V. K., Graham, S. L. Normalization of visual evoked potentials using underlying electroencephalogram levels improves amplitude reproducibility in rats. Invest Ophthalmol Vis Sci. 53 (3), 1473-1478 (2012).
  10. Levkovitch-Verbin, H. Animal models of optic nerve diseases. Eye (Lond). 18 (11), 1066-1074 (2004).
  11. Henry, K. R., Rhoades, R. W. Relation of albinism and drugs to the visual evoked potential of the mouse). J Comp Physiol Psychol. 92 (2), 271-279 (1978).
  12. Murrell, J. C., Waters, D., Johnson, C. B. Comparative effects of halothane, isoflurane, sevoflurane and desflurane on the electroencephalogram of the rat. Lab Anim. 42 (2), 161-170 (2008).
  13. Makela, K., Hartikainen, K., Rorarius, M., Jantti, V. Suppression of F-VEP during isoflurane-induced EEG suppression. Electroencephalogr Clin Neurophysiol. 100 (3), 269-272 (1996).
  14. Boyes, W. K., Padilla, S., Dyer, R. S. Body temperature-dependent and independent actions of chlordimeform on visual evoked potentials and axonal transport in optic system of rat. Neuropharmacology. 24 (8), 743-749 (1985).
  15. Hetzler, B. E., Boyes, W. K., Creason, J. P., Dyer, R. S. Temperature-dependent changes in visual evoked potentials of rats. Electroencephalogr Clin Neurophysiol. 70 (2), 137-154 (1988).
  16. Mitchell, J. The effects of lysolecithin on non-myelinated axons in vitro. Acta Neuropathol. 58 (4), 243-248 (1982).
  17. Meyer, R., et al. Acute neuronal apoptosis in a rat model of multiple sclerosis. J Neurosci. 21 (16), 6214-6220 (2001).
  18. Lachapelle, F., et al. Failure of remyelination in the nonhuman primate optic nerve. Brain Pathol. 15 (3), 198-207 (2005).

Play Video

Citar este artículo
You, Y., Gupta, V. K., Chitranshi, N., Reedman, B., Klistorner, A., Graham, S. L. Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination. J. Vis. Exp. (101), e52934, doi:10.3791/52934 (2015).

View Video