Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Summary

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

Abstract

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases ¹ and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) ².

ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm ³. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 ⁴. Transgenic mice ^4,5 and rats ⁶ carrying mutant human SOD1 genes ^{(G93A, G37R, G86R, G85R)} have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.

Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury ⁷. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons ¹. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion ⁸.

Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest ⁹.

Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise ^10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1^G93A mice and rats, as well as spinal cord injured rats and mice ¹¹.

Protocol

Methods

1. Cell Preparation

As an example, we will describe the procedure for preparing glial progenitor cells ¹² for transplantation because of our experience with this cell type. However, the specifics of the protocol, including medium and use of trypsin for example, will depend on the particular cell type being used for transplantation.

1. Pre-warm all solutions to 37.0°C in water bath.
2. Rinse flask 2X with HBSS. Add 5.0 mL/T-75 flask of 0.05% Trypsin/EDTA. Incubate flask for 3 minutes in 37.0°C incubator. Triturate 3X gently in flask with 5 or 10mL pipette.

Discussion

For studies involving SOD1^G93A mice and rats, age- and sex-match the animals within a group, and distribute animals within the same litter to different groups. It is preferable to use all animals from the same sex for both ALS and SCI models because disease processes may differ between males and females; however, it may also be useful to have enough animals from both sexes to detect possible sex-specific effects, as this phenomenon has been reported with SOD1^G93A rats ¹³ and SOD1^G93A.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HBSS	GIBCO, by Life Technologies	14170
0.05% Trypsin	GIBCO, by Life Technologies	25300
Soybean Trypsin Inhibitor (optional)	Sigma-Aldrich	T-6522
Acepromazine maleate (0.7 mg/kg)	Fermenta Animal Health
Ketamine (95 mg/kg)	Fort Dodge Animal Health
Xylazine (10 mg/kg)	Bayer AG
#11 Feather surgical blade	Electron Microscopy Sciences	72044-11
Cotton-tipped applicators (6 inch)	Fisher Scientific	23-400-101
Rat-toothed forceps	Fine Science Tools	Rat: 11023-15; Mouse: 11042-08
Medium-sized spring scissors	Fine Science Tools	15012-12
Mini spring scissors	Fine Science Tools	15000-10
Rongeur	Fine Science Tools	Rat: 16121-14; Mouse: 16221-14
Microknife	Fine Science Tools	10056-12
Needle holders	Fine Science Tools	12502-14
Suture: 4-0	Vicryl	S-183
Staples: 9 mm	Autoclip	427631
Stapler: 9 mm (Reflex #203-1000)	World Precision Instruments, Inc.	5000344
Warm water pump (T/Pump)	Gaymar Industries	P/N 07999-000
Cyclosporin A: 250.0 mg/5.0 mL ampules	Novartis AG	NDC 0078-0109-01
FK-506	LC Laboratories	F-4900
Rapamycin	LC Laboratories	R-5000
Injector	World Precision Instruments, Inc.	UMP2
Micro 4 Microsyringe Pump Controller	World Precision Instruments, Inc.	UMC4
Micromanipulator	World Precision Instruments, Inc.	Kite-R
10.0 μL Hamilton syringe	Hamilton Co	80030
Hamilton needles: 33-gauge, 45° bevel, 1 inch	Hamilton Co	7803-05
Glass 20.0 μL microcapillary pipettes (optional)	Kimble Chase	71900-20