Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Summary

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Abstract

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.

OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.

All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Introduction

The primary olfactory system is composed of two distinct parts; the olfactory mucosa (OM) in peripheral nervous system and the olfactory bulb (OB) in the central nervous system. The primary olfactory system is characterized by the capacity of the primary olfactory neurons (PON) to self-renew throughout life in mammal species. This ability is made possible due to the presence of neural stem cells in the OM. PON growth and regrowth from OM to OB is facilitated by specialized glial cells called olfactory ensheathing cells (OECs). OECs are neural crest cells which create a favorable microenvironment for neurogenesis of the PON from OM to OB¹. Thereby, OECs can ....

Protocol

1. Primary Cultures of Olfactory Mucosa and Olfactory Bulbs

1. Prior to dissection
   1. Make medium, for 50 ml, by adding 44 ml of calcium-free Dulbecco's Modified Eagle's/Ham's F12 medium (DMEM/F12), supplemented with 5 ml of Fetal Bovine Serum (FBS) and 1 ml of penicillin/streptomycin.
   2. Coat 75 cm² flasks with poly-L-lysine (50 μg/ml, 1.5 μg/cm²), 1 hr at room temperature.
   3. Rinse flasks with PBS 1x.
   4. Store the coated flasks in the fridge for up to 1 week.
2. Dissection
   1. Any method of euthanasia must be approved in advance by the institution's animal care....

Results

Illustrations with control and reinnervated (section/anastomosed) animals have been chosen as results can vary depending on the cellular transplantations performed (OM, OB or OM+OB).

Cell culture
The cells adhere rapidly to the plastic surface and became segregated into parallel swaths of cells that are elongated or tapering, triangular, multipolar, or spindle-shaped (Figures 1A and 1B). After 8 days in vitro flow cytometry analysis shows .......

Discussion

The techniques presented here make OECs a useful model to study cellular transplantations in peripheral nerve injury models. The cell culture protocol is relatively straightforward and can be easily carried out. On the other hand, surgical procedures, in particular section/anastomosis of the RLN, require experience and must be carried out by qualified personnel.

The procedures described in this protocol highlight important factors to focus on in order to obtain the best results possible. First.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM/F12	Invitrogen	E3521T
FBS	Invitrogen	E3387M
Penicilin/streptomycin	Invitrogen	1152-8876
HBSS	Invitrogen	M3467Y
Trypsin-EDTA	Invitrogen	M3513P
Cacodylate	Merck	1.03256.0100
DDSA	Biovalley	00563-450
MNA	Biovalley	00886-450
BDMA	Biovalley	00141-100
Polybed 812	Biovalley	08791-500
PE anti-mouse	BD Bioscience	550589
Matrigel GFR	BD Bioscience	356231
Collagenase A	Roche	10103586001
Mouse anti P75	Chemicon	MAB 365
11.0 Wire	Ethicon	FG 2881
Toluidine Blue	Ral Diagnostics	361590-0025
Centrifuge	Sigma	Sigma 2-16PK
Incubator	Thermo Scientific
Laminar flow hood	Faster	BH-EN 2003 S
Flow cytometer	BD Bioscience	FACSCalibur
Microscope	Zeiss
Videolaryngoscope	Karl Storz Endoskope	Telecam SL NLSC 20212120
Acquisition system	AD Instruments	Powerlab system
Pyramitome Ultramicrotomy System	Leica	Ultracut S
Image analysis system	Explora Nova	Mercator