The Effect Of CSPGs On The Growth Of Primary Afferent Fibers In The Spinal Cord

The primary afferent fibers originated from dorsal root ganglion (DRG) neurons terminate in the spinal cord in a lamina-specific pattern. For example, the nociceptive fibers mainly terminate in the superficial part (lamina I and lamina II) of the dorsal horn (DH), while the proprioceptive fibers terminate in the deep laminae and the ventral horn (VH). This pattern is the basis of spinal cord in the transmission and modulation of sensory information. However, the mechanisms underlying the formation of such a lamina-specific termination pattern are still unclear.It is well known that both chemoattractive and chemorepulsive molecules exist in the nervous system. They guide the propriate extension of axons toward the target area by combining to the growth cone in the axonal ends. Chondroitin sulfate proteoglycans (CSPGs) is one of axon-growth-inhibitory molecules existing in the extracellular matrix (ECM). It can repel the growth of axons in neural development and inhibit the regeneration of axons after spinal cord injury (SCI). When chondroitinase ABC (ChABC) was given to degradate CSPGs in the developmental nervous system such as the optic chiasm, the projection and destination of growing axons were altered. Also in SCI, ChABC treatment in the injuryed sites can increase regenerating ability of axons and improve spinal cord rehabilitation. On the basis of this phenomena, we propose that growth regulation of primary afferent fibers by CSPGs may be involved in the lamina-specific projection and termination of primary afferent fibers in the spinal cord.Using morphological methods and spinal cord slice culture techniques, we investigated the developmental characteristics of primary affernt fibers as well as CSPGs and the temporal-spatial relationships between them. The growth changes of primary afferent fibers after degradation of CSPGs were also observed. Three parts of experiments were included in this study.1. Developmental characteristics of nociceptive and proprioceptive afferent fibers in the mouse spinal cordUsing double immunofluorescent staining method, the growth and termination of nociceptive and proprioceptive afferent fibers in the mouse spinal cord during developmental and early postnatal stages were observed under laser confocal scanning microscope. The results were as follows:(1)CGRP-like immunoreactive (LI) nociceptive fibers first appeared in the superficial part of DH at embryonic (E) 16 day, and entered into the deeper laminae at E17. The fibers increased in both positions at E18, and those in the deep part extended to the superfical laminae. During the early postnatal period (P1 ~ P3), the distribution of CGRP-LI fibers remained unchanged but the intensity of afferent fibers increased and their branching patterns became more complicated. Additionally, projecting fibers that originated from the inner and outer part of DH were also observed at E16 and E17, they elongated ventrally and terminated in the contralateral spinal cord by crossing the posterior commissure. The number of fibers gradually increased with time till birth.(2)PV-LI proprioceptive afferent fibers first entered the gray matter by crossing the dorsal funiculus at E15. They went though the inner part of intermediate gray matter (IG) and terminated mainly both in the inner and outer part of VH. Some fibers terminated in the IG. The distribution characteristics of fibers remain unchanged in the spinal cord besides the increase in density. At E17, the terminals of PV-LI fibers began to form close contacts with motoneurons in the VH and the number of these contacts increased with age.The results of this part indicate that the somatotopic organization of both nociceptive and proprioceptive afferents in the spinal cord are established during the late embryonic and early postnatal stages. This spatial-temporal specificity in the pattern of primary afferent fibers help us to understand the development of sensory transmission and modulation in the spinal cord in more details.2. Developmental changes of CSPGs and its distribution relationship with nociceptive and proprioceptive afferent fibers in the mouse spinal cord Using immunofluorescent staining method, the developmental changes of CSPGs and its distribution relationship with nociceptive and proprioceptive afferent fibers in the mouse spinal cord were observed under laser confocal scanning microscope. The results were as follows:(1) CSPGs first appeared in the VH at E12, and then showed an increased in density with time, also spread toward the DH. At E17 and E18, CSPGs were well seen in the deep part of DH, but poorly in the superficial part. In the whole spinal cord, CSPGs showed a ventral-toward-dorsal decreasing tendency. At P1, the distribution pattern of CSPGs did not change, but showed a decrease in density.(2) At E16 and E17, CGRP-LI nociceptive fibers were mainly seen in the superficial part of DH (lamina I and lamina II), where CSPGs were not seen. At E18 and P1, the laminae I and II were seen with increased amount of CGRP-LI nociceptive fibers, some of which extended and terminated in the lamina V of DH. But CSPGs were never seen beyond the lamina III of DH at the same time. Although they showed some coexistence in the lamina V, it was hard to observe an obvious ventral-toward-dorsal decreasing tendency of CSPGs at this time point.(3) At E16, E17 and P1, the PV-LI fibers entered the spinal cord from dorsal funiculus and terminated in the medial and lateral part of VH by extending ventrally through the inner part of IG. CSPGs showed a decreased intensity from VH toward DH in the spinal cord at the same time. PV-LI fibers have a dense distribution in the VH, where CSPGs expressed highly.The results of this part revealed that CSPGs distributed with graded intensity in the spinal cord during development. The distribution of CSPGs were overlapped with that of prociopreceptive fibers, but not or few with that of nociceptive fibers, which suggest that CSPGs may have selective inhibitory effects on the growth and termination of primary afferent fibers in the spinal cord.3. Effects of CSPGs on the growth of primary afferent fibers in the mouse spinal cordUsing spinal cord slice culture technology and immunofluorescent staining method, growth alteration of primary afferent fibers particularly the CGRP-LI nociceptive fibers after degradation of CSPGs in the development mouse spinal cord were observed under laser confocal scanning microscope. The results were as followed:(1) After sparately treated with 0.1 U/ml and 0.5 U/ml ChABC, the distribution pattern of CSPGs in the mouse spinal cord slice at E18 ~ P0 were both not influenced significantly, but their expression intensity decreased and the ventro-dorsal degradation pattern became obscure. Such changes were more obviously at higher concentration of ChABC (0.5 U/ml).(2) After treated the spinal cord slice at E18 ~ P0 with ChABC at a final concentration of 0.1 U/ml, CGRP-LI fibers in the superficial layers (lamina I and lamina II) of DH and their branches were seen to extend ventrally instead. When the concentration of ChABC increased to 0.5 U/ml, the above changes were more obvious. Those fibers, which originally terminated and branched in the superficial part of DH, almost totally extended ventral-laterally, and the distribution of fibers in the deep layer (lamina V) changed more greatly.(3) In the spinal cord slice at E18 ~ P0, the distribution pattern of PV-LI proprioceptive fibers were observed unchanged after 0.1 U/ml and 0.5 U/ml ChABC treatment. PV-LI fibers remain terminated mainly both in the inner and outer part of VH by elongating though the inner part of IG.These results indicate that ChABC can degradate CSPGs in the mouse spinal cord during development and only change the growth pattern of nociceptive fibers. This show that CSPGs have selective repel effects on the growth of primary afferent fibers and these effects are proportional to the concentration of CSPGs.