Degeneración de los terminales aferentes primarios de rata luego de lesión extensa por avulsión del plexo braquial

Vilma Muñetón-Gómez, Julian Scott Taylor, Sharon Averill, John V. Priestley, Manuel Nieto-Sampedro
2004 Biomédica: revista del Instituto Nacional de Salud  
Important breakthroughs in the understanding regeneration failure in an injured CNS have been made by studies of primary afferent neurons. Dorsal rhizotomy has provided an experimental model of brachial plexus (BP) avulsion. This is an injury in which the central branches of primary afferents are disrupted at their point of entry into the spinal cord, bringing motor and sensory dysfunction to the upper limbs. In the present work, the central axonal organization of primary afferents was examined
more » ... in control (without lesion) adult Wistar rats and in rats subjected to a C3-T3 rhizotomy. Specific sensory axon subtypes were recognized by application of antibodies to the calcitonin gene-related peptide (CGRP), the P2X3 purinoreceptor, the low-affinity p75-neurotrophin receptor and the retrograde tracer cholera toxin subunit β (TCβ). Other subtypes weres labeled with the lectin Griffonia simplicifolia IB4. Using immunohistochemistry and high resolution light microscopy, brachial plexus rhizotomy in adult rats has proven a reliable model for several neural deficits in humans. This lesion produced different degrees of terminal degeneration in the several types of primary afferents which define sub-populations of sensitive neurons. Between the C6 and C8 levels of the spinal cord" deafferentation was partial for peptidergic GCRP-positive fibers, in contrast with elimination of non peptidergic and myelinated fibers. Dorsal rhizotomy has provided an adequate experimental model to study sensory alterations such as acute pain and allodynia as well as factors that affect regeneration into the CNS., Therefore, the differential deafferentation response must be considered inr the evaluation of therapies for nociception (pain) and regeneration for brachial plexus avulsion. The anatomical diffierences among the primary afferent subtypes also affect their roles in normal and damaged conditions. Key words: primary afferents, cervical spinal cord, dorsal root avulsion, lectin Griffonia simplicifolia (IB4), cholera toxin subunit b (CTβ), calcitonin gene-related peptide (CGRP), purinoreceptor (P2X3). Degeneración de los terminales aferentes primarios de rata luego de lesión extensa por avulsión del plexo braquial El uso de las neuronas sensoriales primarias ha aportado avances en el entendimiento de las razones por las cuales falla la regeneración cuando el sistema nervioso central (SNC) es dañado. La rizotomía dorsal se puede usar como un modelo experimental de las lesiones por avulsión del plexo braquial, una lesión en la cual son desprendidas, en su punto de entrada en la médula espinal, las ramas centrales de los aferentes primarios causando una disfunción motora y sensorial grave e irreversible del miembro superior. En el presente trabajo, se examinó la organización central de los aferentes primarios en ratas Wistar adultas. Éstas fueron divididas en controles normales no lesionados y en animales rizotomizados entre los niveles cervical 3 y torácico 3 (C3-T3). Se estudió la deaferentación de los subtipos de axones sensoriales utilizando anticuerpos específicos contra el péptido relacionado con el gen de la calcitonina (CGRP), el receptor purinérgico (P2X3), el receptor de baja afinidad p75 para el factor de crecimiento nervioso (NGF) y contra la subunidad β de la toxina de cólera (TCβ). Otro subtipo fue marcado con la lectina Griffonia simplicifolia IB4. La inmunohistoquímica y la microscopía 184 óptica de alta resolución demostraron que el modelo animal de rizotomía completa del plexo braquial reproduce diversos déficit observados en las lesiones humanas. Esta lesión produce diferentes grados de degeneración terminal entre los diversos tipos de aferentes primarios que definen subpoblaciones de neuronas sensoriales. En los niveles de la médula espinal estudiados (entre C6 y C8), la deaferentación fue parcial para las fibras peptidérgicas GCRPpositivas, en contraste con la eliminación de las fibras no peptidérgicas y las mielinizadas. La rizotomía dorsal es un modelo experimental apropiado para estudiar las alteraciones sensoriales como el dolor agudo y la alodinia, así como los factores que podrían afectar la regeneración en el SNC. Por tanto, la respuesta de deaferentacion diferencial debe ser tenida en cuenta para la evaluación de terapias antinociceptivas y regenerativas tras la avulsión del plexo braquial. Se discute la anatomía de los subtipos de aferentes primarios y su papel en condiciones normales y después de la lesión. Palabras clave: aferentes primarias, médula espinal cervical, avulsión de la raíz dorsal, lectina Griffonia simplicifolia (IB4), subunidad b de la toxina de cólera (CTb), péptido relacionado con el gene de la calcitonina (CGRP), purinorreceptor (P2X3) The cervico-thoracic zone of the spinal cord receives the projections of the nerves corresponding to the upper limbs and forms the brachial plexus, between C3 and T3 (1-3). Lesions produced by brachial plexus avulsion are frequently caused in adult humans during motorcycle, sports and industrial accidents, and in children by hyperstretching of the angle between the head and neck. The experimental lesion of dorsal rhizotomy and avulsion models in rats reproduce the damage produced by human avulsion (4-6). Depending on the extent of the lesion, consequences include severe motor and sensory deficit and upper limb paralysis, reflex loss, hypersensitivity, acute pain and allodynia and, in the most dramatic cases, self-mutilation (7) . Primary afferent neurons transmit sensory information from tissues and organs. Their cell bodies lie within the dorsal root ganglia (DRG) and are conveniently located for the study of the mechanisms controlling regenerative failure because they have axonal branches both in the central nervous system (CNS) and in the peripheral nervous system (PNS) (8). Following a lesion, the central and peripheral projections show different regenerative capacities. While the peripheral axons show a strong regenerative ability that is supported by their associated Schwann cells (9), the damaged central afferents do not regenerate into the spinal cord (10,11). Dorsal root section (rhizotomy) results in a fast and complete degeneration of the central terminal roots of the axotomized afferents (12). Thus, the primary afferent system has provided a useful model to study the mechanisms preventing regeneration in the central nervous system. Primary afferent neurons feature a high-level organization according to their chemical phenotype, conduction velocity, axonal diameter, peripheral receptors, presence or absence of myelin, and axonal arborization pattern within the dorsal horn (13-15). The afferents are classified into two main groups of fibers according to their axon diameter: large (A fibers) which have thick myelinated axons, and small which have nonmyelinated axons (C fibers) or thin-caliber myelinated axons (A delta fibers). Large and medium-sized DRG neurons may be identified by their content of phosphorylated heavy chain (200 kd) neurofilament (16, 17) . These neurons also express the GM1 ganglioside and give rise to myelinated axons. The beta subunit of cholera toxin (CTβ), which binds to GM1 and is then internalized by endocytosis, can be injected into peripheral nerves and used to transganglionically label A beta terminals in laminae III and IV of the spinal cord and A delta terminals in lamina I (18,19) . The small C fiber DRG cells have been classified on the basis of their function and phenotype (20) 185 Biomédica 2004;24:183-93 PRIMARY AFFERENT TERMINALS AND BRACHIAL PLEXUS AVULSION into two groups: peptidergic and non-peptidergic. The C fibers that express neuromodulatory peptides (e.g., substance P; SP, calcitonin gene related peptide, CGRP) project to lamina I and lamina II outer of the dorsal horn (21-23). Many of these peptidergic fibers also express the p75 neurotrophin receptor (24). The C fibre nonpeptidergic DRG cells project to lamina II inner of the dorsal horn. These fibers express a glycoprotein that binds the lectin Griffonia simplicifolia IB4 and may also be identified by their fluoride-resistant acid phosphatase activity (25-27). Most of them express the ionotropic purinergic receptor for ATP, P2X3 (28,29). Finally, there is a small population of DRG neurons that bind IB4 and also contain CGRP (24,30). Unilateral dorsal rhizotomy leads to neuroanatomical (31), propioceptive (5), sensory and motor alterations (2, 31), in the forepaw of adult rats. The objectives of present work are: 1) using rhizotomies, to establish an animal model with dorsal horn afferent elimination which allows the evaluation of regenerative and reparative therapies for brachial plexus avulsion; 2) by inmunohistochemistry, to evaluate the effect of complete rhizotomies of brachial plexus over several types of primary afferents. The present work shows how rhizotomy in adult rats reproduces the injuries reported in humans following brachial plexus avulsion. The anatomical results, and the role of the different types of primary afferents in normal and injured conditions, will be discussed further. Methods Surgery Adult male Wistar rats (250-300 g) were obtained from the Instituto Cajal and maintained in standard conditions with access to food and water ad libitum. The experimental protocols adhered to the recommendations of the European Council and Spanish Department of Health for Laboratory Animals and were approved by the Animal Care Committees of our institutions. A total of 24 rats were divided into controls (n=8) and rhizotomy (n=16) animals. Rhizotomized animals were anaesthetized with sodium pentobarbital (Nembutal©, Abbott Laboratories; 50 mg/kg i.p.) and Rompún© (2-5,6-dihidro-4H-1,3-thiazine hydrochloride, Bayer; 3 mg/kg i.p.). The back of each animal was shaved and disinfected with polividone iodine (Asta Medica, Madrid). After opening the muscular layers and fascia with a No. 11 scalpel (Aesculap AG & Co. Kg), an extensive cervico-thoracic hemilaminectomy was performed under aseptic conditions, opening the dura mater with fine iridectomy scissors (Fine Science Tools). After topical application of tetracaine (0.5%, Alcon Cusi SA, El Masnou, Barcelona), the roots between C3 and T3 were identified and cut more than 1mm away from the DREZ using fine iridectomy scissors. The spinal cord was then covered with Gelfoam (Upjohn Co., Kalamazoo, MI), soaked in sterile saline, and the muscle and skin layers were closed together with chromic gut suture and surgical clips, respectively. The animals were maintained in a warm environment until full recovery from anesthesia. In order to evaluate the effect of the rhizotomy on the A beta and A delta sensory afferents, cholera toxin ß (CTß) (33) was used as a transganglionic tracer in three rats of both groups, the control and the rhizotomized animals. In these cases the animals were reanaesthetized 1-3 days prior to perfusion (see below) and 4 µl of 1% CTß was injected using a Hamilton syringe, after which the muscle and skin layers were closed and animals were allowed to recover. Histology After survival periods of 3 weeks, all the animals were deeply anaesthetized with sodium pentobarbital (Nembutal©, Abbott Laboratories; 60 mg/kg) and perfused through the ascending aorta with 200 ml of 0.01 M phosphate buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The spinal cord was dissected out, postfixed in 4% paraformaldehyde for 1 to 2 hours, and cryoprotected in 25% sucrose in PB for 1-2 days. Tissue was included in Tissue-tek O.C.T. (Sakura Finetek), frozen in solid CO 2 and transverse 30 µm cryostat sections were recovered throughout C6-C8 on electrostatically-charged glass slides (Merck). Inmunohistochemistry Sections were processed for immunofluorescence using a variety of antisera in order to detect 186
doi:10.7705/biomedica.v24i2.1264 fatcat:uyik6xktfbhi3pagepzevuzn24