ABSTRACT
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 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 beta (TCbeta ). 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.
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 ®de la toxina de cólera (TCbeta ). Otro subtipo fue marcado con la lectina Griffonia simplicifolia IB4. La inmunohistoquímica y la microscopía 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.
Subject(s)
Animals , Male , Rats , Brachial Plexus/injuries , Disease Models, Animal , Neurons, Afferent/pathology , Axons , Neurons, Afferent/cytology , Rats, Wistar , RhizotomyABSTRACT
Objective:To lay a foundation of expressing VCTB fusion protein and preparation the oral vaccine for prophylaxis and therapy of H.pylori infection,the prokaryotic expression vectors contained the fusion gene vacA toxic subunit of H.pylori and cholera toxin subunit B(ctxB)was constructed.Methods:The high homogeneity gene fragment was found in comparing VacA toxic subunit gene of domestic typical H.pylori strains.The primers were designed according to vacA and ctxB sequences in the gene bank.VacA toxic subunit gene was amplified by PCR as the template of DNA genome of H.pylori and cloned into plasmid pQE30,which was plasmid pQE30-vacA.The ctxB gene was amplified by PCR as the template of pET32(?)+-ctxB plasmid.The purified ctxB gene was inserted into pQE30-vacA to construct expressing plasmid pQE-vctB of containing vacA and ctxB genes.pQE-vctB was transformed into prokaryotic E.coli Top10.The recombinant plasmid of bacteria cultivated was extracted and purified,and cutted with the incision enzyme to evaluated.Results:The sequence of vacA gene amplified was about 723 bp and the sequence of ctxB gene was about 372 bp.They were consistented with the anticipated length of the DNA.The expressing plasmid pQE-vctB was conformitied with objective gene inserted into pQE30.vctB fusion gene sequenced as 1 092 bp by the sequencing was conformitied with the genebank,and encodes polypeptides of 364 amino acid residues.Conclusion:The prokaryotic expression vectors contained vacA and ctxB fusion gene was constructed successfully and lay a foundation of expression VCTB fusion protein.