Substance P immunoreactivity in the lumbar spinal cord of the turtle Trachemys dorbigni following peripheral nerve injury

Abstract

Immunoreactive substance P was investigated in turtle lumbar spinal cord after sciatic nerve transection. In control animals immunoreactive fibers were densest in synaptic field Ia, where the longest axons invaded synaptic field III. Positive neuronal bodies were identified in the lateral column of the dorsal horn and substance P immunoreactive varicosities were observed in the ventral horn, in close relationship with presumed motoneurons. Other varicosities appeared in the lateral and anterior funiculi. After axotomy, substance P immunoreactive fibers were reduced slightly on the side of the lesion, which was located in long fibers that invaded synaptic field III and in the varicosities of the lateral and anterior funiculus. The changes were observed at 7 days after axonal injury and persisted at 15, 30, 60 and 90 days after the lesion. These findings show that turtles should be considered as a model to study the role of substance P in peripheral axonal injury, since the distribution and temporal changes of substance P were similar to those found in mammals. Key wordsPeripheral nerve injury may result in significant changes in neuropeptide production and the development of neuropathic pain behavior (1). In rats, peripheral nerve sectioning leads to a marked quantitative decrease in the nerve of substance P, a peptide of 11 amino acids that is present in small primary afferents and plays an important role in pain sensation (2,3).The chemical structure of substance P is highly conserved in non-mammalian species, and is also found in turtles, animals considered phylogenetically related to the extinct theropsids from which mammals arose (4). Previous studies demonstrated substance P immunoreactivity in the turtle central nervous system (5,6); however, the effects of peripheral axotomy on substance P distribution have not been studied. Therefore, the aim of this study was to determine the effects of sciatic nerve transection on substance P immunoreactive fibers in the lumbar spinal cord of the turtle Trachemys dorbigni.Under ether-induced anesthesia (7), the right sciatic nerve was exposed and transected approximately 5 mm distal to the sciatic notch. In this nerve transection, a 2-mm segment of the nerve was removed to ensure that the transection was complete. Groups of three turtles of both sexes, weighing 300-400 g, were sacrificed 7, 15, 30, 60 and 90 days later. On the final day of the experiment, both operated and control turtles were anesthetized (25 mg/kg Thionembutal, intraperitoneally) and perfused through the heart with cold saline solution followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The lumbar spinal cord was quickly dissected out, immersed in the same fixative for 2 h and then cryoprotected in 15 and 30% sucrose solutions in phosphate buffer at 4ºC. Coronal serial sections (50 µm) were obtained with a cryostat (Leitz) and collected in cold phosphate-buffered saline (PBS). The sections were then treated with 3% hydrogen peroxide in 10% methanol for 30 min, washed wit...

Immunohistochemical and hodological characterization of calbindin‐D28k‐containing neurons in the spinal cord of the turtle, Pseudemys scripta elegans

“…This similarity supports that synaptic field II of turtles is homologous to the substantia gelatinosa of mammals (Trujillo-Cenóz et al, 1990). Similarly, synaptic field Ia was considered analogous to Lissauer's tract in mammals (Partata et al, 2003;Trujillo-Cenóz et al, 1990) but according to the present results combining the labeling of primary afferents and CB it is clarified that Lissauer's tract courses adjacent to synaptic field Ia. The intense CBir fibers in the superficial dorsal horn of the turtle do not appear to originate from primary afferents, as demonstrated in the double labeling experiments.…”

Time course of substance P expression in dorsal root ganglia following complete spinal nerve transection

“…SP is a neuromodulator present in 6–20% of the DRG neuronal population, usually limited to small sensory neurons (our findings; Otsuka and Yoshioka,1993). Peptide content and gene expression products in DRG neurons, axons, and projections into the dorsal horn are generally down‐regulated when assayed 1 week or more after peripheral nerve transection or tight ligation (Ahmed et al,1995; Zhang et al,1995a,b; Ji et al,1996; Rydh‐Rinder et al,1996; Sterne et al,1998; Ma and Bisby,1998a; Mohiuddin et al,1999; Antunes Bras et al,1999; Sondell et al,1999; Honore et al,2000; Siri et al,2001; Xiao et al,2002; White and Kocsis,2002; Wang et al,2002; Partata et al,2003; Hofmann et al,2003; Valder et al,2003; Sanderson et al,2004; Swamydas et al,2004; for earlier studies see also Hokfelt et al,1994). Nerve compression also modulates the content of SP and gene expression in primary sensory neurons (Bisby and Keen,1986; Cameron et al,1997; Sondell et al,1999; Lee et al,2001; Wong and Tan,2002; Kobayashi et al,2004; Swamydas et al,2004).…”