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 be found in OM and in OB constituting different subpopulations of cells^2,3. The different properties of OECs have lead scientists to use them for cellular transplantations in several nervous system lesion paradigms⁴. Indeed, OECs produce growth factors, reduce glial scaring, promote axonal regrowth, and can freely intermingle with astrocytes^5,6. However, the vast majority of these studies are based on spinal cord injury (SCI); few of them have used OECs after peripheral nerve injury (PNI)^7,8.

Although the peripheral nervous system has a great capacity to regenerate after nerve injury, complete sections induce aberrant axonal regrowth. Indeed, after complete transections of the facial or the recurrent laryngeal nerves (RLN) misrouted axons cause muscular cocontractions called synkinesis. Therefore it is of primary importance to propose a model of PNI to not only quantify axonal regrowth but also to evaluate the efficiency of the muscular contractions. In the literature the most common model described is based on facial nerve lesion^9,10. In this model, functional evaluations are based on the recovery of whisker movements¹⁰. However it is complicated to demonstrate the efficiency of the movements and to discriminate the muscular cocontractions phenomena. We propose here a model based on a RLN lesion. This model allows the evaluation of not only axonal regrowth and movements of the vocal cords but also the efficiency and functionality of these movements after cellular transplantations^11,12. This protocol provides a step by step procedure to culture and transplant OECs from OM and OB in an RLN section/anastomosis model and to evaluate animals after surgery.

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 and use committee and carried out by qualified personnel.
   2. Choose inbred rats such as Fischer rats.
   3. After the rat has been confirmed to have entered deep anesthesia with sodium pentobarbital or other injectable forms of anesthesia such as ketamine/xylazine, decapitate him and remove the skin.
   4. Open the skull from the anterior to the posterior part.
   5. Remove the OB taking care to avoid the meninges.
   6. In the same rat, open the nasal cavity sagittaly.
   7. Remove the OM taking care to avoid the respiratory mucosa. Note that the OM epithelium can be easily identified by its yellowish color.
   8. Further detailed protocols about dissection of OM can be find in a previous J. Vis. Exp. publication¹³.
3. Culture
   1. Place OB in Hank's buffered salt solution (HBSS) containing 0.1% trypsin for 45 min at 37 °C.
   2. Place OM in HBSS containing 0.05% collagenase A for 45 min at 37 °C.
   3. Stop enzymatic digestion by adding 2 ml of warm medium.
   4. Wash the cells with medium and centrifuge them at 300 x g for 3 min.
   5. Triturate samples of 2 ml of medium using a P1000 pipette until a homogenous cell suspension is obtained.
   6. Plate the cells in the precoated flasks (5-10 x 10⁶ cells/flask) with 25 ml of warm medium.
   7. Incubate and culture the cells at 37 °C with 5% CO₂.
   8. Change half of the medium every 2 days.
   9. After 6-8 days in vitro, check the proportion of p75 positive cells on small cell samples, assumed to be OECs in rodents, by flow cytometry or by immunocytochemistry. For these analyses, other markers can be studied such as GFAP and S100β.
4. Flow cytometry analysis
   1. Collect the cells as described in "cellular transplantation" (section 2.2 below).
   2. Incubate them with p75 primary antibody (1:100) during 15 min at 4 °C.
   3. Wash the cells with 2 ml of PBS-EDTA and centrifuge them at 300 x g for 3 min.
   4. Incubate the cells with PE conjugated anti-mouse secondary antibody during 15 min at 4 °C in the dark (1:200).
   5. Wash the cells with 2 ml of PBS-EDTA and centrifuge them at 300 x g for 3 min.
   6. Resuspend the cells in 500 μl of PBS-EDTA.
   7. Analyze 10-30 x 10³ cells with flow-cytometer.
5. Immunocytochemistry analysis
   1. Plate the cells in 6-well plates precoated for 48 hr as described in "culture" (section 1.1.2 below).
   2. Wash the cells with PBS.
   3. Fix the cells with PFA 4% for 15 min at RT.
   4. Wash the cells with PBS.
   5. Incubate the cells with PBS/Triton X100 (0.1%) for 15 min at RT.
   6. Wash the cells with PBS.
   7. Incubate the cells with primary antibodies (1:200): p75, S100β and GFAP overnight at 4 °C.
   8. Wash the cells with PBS.
   9. Incubate the cells with the secondary antibodies (1:500) during 1 hr at RT in darkness.
   10. Wash the cells with PBS.
   11. Incubate the cells with Hoechst 10 min at RT in darkness.
   12. Wash the cells with PBS.
   13. Analyze the cells using a fluorescent microscope.
6. GFP labeling cells
   1. Five days before transplantation, infect OB and OM cultures overnight with lentiviral vector harboring enhanced GFP (multiplicity of infection: 20).
   2. Before transplantation check on small samples the rate of GFP positive cells by flow-cytometry as described previously in "culture" (section 1.1).

2. Surgery and Transplantation

1. Recurrent nerve anastomosis
   1. All experiments with animals must be approved in advance by the institution's animal care and use committee and carried out by qualified personnel.
   2. Prior to surgery autoclave all instruments and maintain sterility throughout the surgical procedures.
   3. Anesthetize rat by intraperitoneal injection of ketamine hydrochloride (12.5 mg/kg) and chlorpromazine hydrochloride (0.625 mg/kg). Assess adequacy of anesthesia by toe pinch prior to surgery.
   4. Place the rat in dorsal decubitus.
   5. Perform a 2 cm vertical medium cervical cutaneous incision with a scalpel.
   6. Perform a vertical incision of the infrahyoid muscles following the medial white line.
   7. Expose larynx by using autostatic retractor between infrahyoid muscles.
   8. Expose the RLN.
   9. Under microscopic control, cut the nerve fully with microscissors at the level of the seventh tracheal ring.
   10. Perform anastomosis using one point of 11.0 suture under microscopic control. This surgery leads to a specific motor denervation of the larynx.
2. Cellular transplantation
   1. Just before transplantation, remove the cells from the dishes using 0.05% trypsin EDTA for 5 min at 37 °C.
   2. Stop enzymatic digestion by adding 2 ml of warm medium.
   3. Centrifuge at 300 x g for 3 min.
   4. Under a microscope count the cells using a hemocytometer and adjust the cellular concentration according to specific protocol. In this model, desired concentrations were between 1.2-6 x 10⁶ cells in 30 μl of DMEM/F12.
   5. Make cellular preparation with 1:2 mix (30 and 60 μl in this experiment) of DMEM/F12-Matrige l in 1.5 ml tube before grafting and place it on ice. To avoid unspecific effects, use growth factor reduced Matrigel.
   6. Just prior to transplantation hold the tube in your hand for a few seconds to warm up the Matrigel-medium mix.
   7. Depose the mix on the anastomosis site using a pipette and wait until the mixture self-assembles on the injured nerve.
   8. Close the muscles and skin with a 4.0 suture.
   9. House the rats individually and keep under a heating lamp for 24 hr.

3. Evaluation

1. Laryngoscopy
   1. Anesthetize rats by intraperitoneal injection of ketamine hydrochloride (12.5 mg/kg) and chlorpromazine hydrochloride (0.625 mg/kg). Confirm rats are anesthetized with a toe pinch prior to proceeding with protocol.
   2. Place the rat in dorsal decubitus.
   3. Insert a 30° videolaryngoscope adjusted to provide the best view of the larynx. Determine anatomic landmarks to reproduce the same view for each recording.
   4. Record vocal cord movements using the videolaryngoscope (5-10 sequences/animal). For each animal select three sequences of successive maximal adduction and maximal abduction.
   5. Analyze laryngeal function using image analysis software. Determine absolute angular movement by measuring the difference between movement of maximal abduction and maximal adduction of the vocal cords. Determine dynamic score by measuring the movement amplitude. Determine functional score by measuring the synkinesis and paradoxal movements.
2. Electromyography (EMG)
   1. Place an electrode on the hamstring muscles of the rat for electrical isolation.
   2. Expose larynx and trachea by opening skin and muscles as described previously in "recurrent nerve anastomosis" (section 2.1).
   3. Expose the cricothyroid cartilage.
   4. Open the cricothyroid cartilage to expose the posterior cricoarytenoid (PCA) muscle using microscissors.
   5. Introduce a monopolar needle electrode (38 x 0.45 mm) in the PCA muscle.
   6. Record the electrical muscular activity of the PCA muscle using an acquisition system during spontaneous ventilation.
   7. Analyze the muscular activity in terms of richness and synchronization with the respiration. To analyze EMGs, assign a qualitative score from 0-3 using the following scale: 0: unrhymed tracing, without increase during inspiration, 1: rhymed tracing with inspiratory increasing, but poor tracing (neurogen), 2: rhymed tracing with richer activity, 3: rhymed tracing, very rich, constituting an interference pattern, similar to a maximal intentional activity.
   8. Expose the vagus nerve to measure latency and potential duration. The vagus nerve is chosen as the site of stimulation because RLN can be damaged during stimulation due to its small size.
   9. Stimulate the vagus nerve with an electrode.
   10. Record electrical signals in the PCA muscle. Measure latency and potential duration. If possible use an acquisition module to record muscular activity, latency and potential duration such as Powerlab system.
3. Histology
   1. Euthanize the animal using pentobarbital overdose.
   2. Remove the distal stump of RLN under microscopic control. The distal part of RLN is defined as the part between anastomosis and larynx. Note that it is possible to find the anastomosis site several weeks after surgery because the 11.0 suture is a nonabsorbable suture.
   3. Place the distal portion of RLN in 2% glutaraldehyde with 0.1 M phosphate (pH=7.3) for 2 hr at 4 °C.
   4. Carefully orient the samples to realize coronal sections.
   5. Wash samples in phosphate buffer.
   6. Cut in smaller segments (3-4 mm length) and postfix for 60 min at room temperature in 1% cacodylate buffered OsO₄ and dehydrated with ethanol.
   7. Orient samples in silicone molds and embedded in resins consisting in POLYBED 812, DDSA and MNA, to which was added 2% of the accelerator BDMA.
   8. Make semi-thin transverse sections (1μm-thick) using a Pyramitome Ultramicrotomy System.
   9. Stain with 0.1% Toluidine Blue in 1% sodium tetraborate for 75 sec at 70 °C.
   10. Analyze RLN samples with a counting analysis system. Count the number of fibers and measure myelinated nerve fiber profiles by determining the outer and inner boundary of the myelin sheath.
4. Transplantation
   1. Prepare cells for transplantation and transplant the cells as previously described in "cellular transplantation" (section 2.2).
   2. Analysis
   3. Euthanize the animal using pentobarbital overdose. Note that fixation of the animals can be performed using intracardiac injection of PFA 4%.
   4. Remove around 2 cm of RLN. Use proximal and distal parts of the RLN.
   5. Fix RLN sample in liquid nitrogen.
   6. Store samples at -80 °C.
   7. Cut samples longitudinally using cryostat (10-20 μm thick).
   8. Analyze samples under fluorescent microscope to detect GFP positive cells. Note that anti-GFP antibody can be used to increase fluorescence emission and to perform costainings.

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