| Objective: To observe the gene expression patterns ofβ-1, 4-Galactosyltransferase I and V in rat models of spinal cord development,injury and CFA-induced inflammatory pain as well as to approach their potential biological roles in central nervous development, injury and pain process or modulation.Methods: Sprague-Dawley (SD) rats were subjected to different development stage, spinal cord contusion or transection injury. Gene expression patterns forβ-1, 4-Galactosyltransferase I and V were detected in these modles by Real-time fluorescence quantitative PCR as well as in situ hybridization. More measures such as Western blot, Lectin-fluorescent staining with RCA-I, immunofluorescence and immunoprecipitation were taken besides Real-time fluorescence quantitative PCR and in situ hybridization to study the expression patterns ofβ-1, 4-Galactosyltransferase I and V. Additonally, thermal hyperalgesia was induced by an injection of complete Freund's adjuvant (CFA) into one hindpaw of the SD rat, and the changes in mRNA forβ-1,4-Galactosyltransferase I and V were also examined in the corresponding DRG and lumbar spinal cord.Results: (1) We examined the spatiotemporal expression ofβ-1,4-GalT-I in the lumbar spinal cord at various developmental stages. Temporally, Real-time PCR analysis revealed that theβ-1,4-GalT-I gene was expressed mainly in mid-embryonic stage, peaked at the mid-embryonic day 18 (E 18d), and then decreased to reach an adult level. Spatially, β-1, 4-GalT-I was located in NeuN-positive neurons at P 18. In addition, the enhanced immunoprecipitations of neural cell adhesion molecule (NCAM) were detected in E 18 d developing spinal cord.(2) Real-time PCR revealed thatβ-1,4-GalT-I mRNA peaked at 1 d after spinal cord contusion. In situ hybridization indicated thatβ-1, 4-GalT-I mRNA was adjacent to the center of injury. Double immunofluorescent staining showed thatβ-1,4-GalT-I mostly overlapped with ED1-positive macrophages 1 d after SCI, partly colocalized with microglia, neutrophils and a few with oligodendrocytes and astrocytes. The result of Lectin-fluorescent staining with RCA-I was similar to that of double immunofluorescent staining. Terminal galactosylation of E-selectin underwent obvious changes between sham and 3 d after SCI by immunoprecipitation of E-selectin.(3) We examined the spatiotemporal expression ofβ-1,4-GalT-V in the lumbar spinal cord at various developmental stages.β-1,4-GalT-V mRNA was predominantly expressed in neurons of gray matter at 3-day newborn rat (P3d) and the number ofβ-1,4-GalT-V-positive cells increased significantly. The gene expression ofβ-1, 4-GalT-V suffered alterations after rat spinal cord injury (SCI). Spinal cord contusion (SCC) and spinal cord transection (SCT) model were established in Adult Sprague Dawley (SD) rats. In SCC model,β-1,4-GalT-V mRNA was increased significantly at 8 h and reached the peak at 1 d, recovered to the baseline level at 14 d. In the rostral side of SCT model, the relative amounts ofβ-1,4-GalT-V mRNA showed noticeable changes with time points post injury, the increase ofβ-1,4-GalT-V mRNA level was prominent at 8 h. In the caudal side of SCT model, the transcript ofβ-1, 4-GalT-V displayed a marked increase at 1 d and then reduced to the minimum level at 14 d after injury. In situ hybridization showed thatβ-1, 4-GalT-V mostly overlapped with ED-1-positive macrophages 1 d after SCI, partly colocalizaed with oligodendrocytes and microglia. Histological studies showed thatβ-1,4-GalT-V mRNA was also localized in where substance P was positive, and several of which co-expressed with IB4, a marker for subpopulations of nociceptive and thermoreceptive neurons.(4) Thermal hyperalgesia was induced by CFA injection in the ipsilateral hindpaw of the SD rat, which was predominant in 4–6 h and last to 72 h. However, this did not happen in the contralateral side or in the normal saline (NS) group. In this inflammatory pain model,β-1,4-GalT-I mRNA was significantly increased in the ipsilateral corresponding DRG as early as 1 h post injection, and reached its peak at 1 d. While in the lumbar SC, the expression ofβ-1, 4-GalT-I mRNA was not abundant after CFA injection. In contrast,β-1, 4-GalT-V mRNA markedly peaked 1 d in ipsilateral DRG. In the lumbar SC,β-1, 4-GalT-V mRNA was increased as early as 1 h post injection, and reached high level at 4 h and 2 d with a peak at 1 d. Histological studies showed thatβ-1, 4-GalT-V mRNA was localized in small- and medium-diameter neurons of DRG, several of which co-expressed with IB4. Abundant mRNA signals ofβ-1, 4-GalT-V were showed in superficial laminae of the spinal dorsal horn (I and II), which predominantly receive input from nociceptive and thermoreceptive primary afferents.Conclusions: (1) During spinal cord development, the spatio-temporal expression ofβ-1, 4-GalT-I were abundant in mid-embryonic day 18 (E 18d), for this function ofβ-1, 4-GalT-I is partially mediated by NCAM, suggestingβ-1,4-GalT-I might participate in neuroal development of rat spinal cord.(2)β-1,4-GalT-I was dominantly expressed by macrophages, microglia and neutrophils after traumatic insults of the spinal cord in vivo. One of the functions ofβ-1,4-GalT-I was most likely to participate in inflammation of adjacent damaged cells during the early stage of traumatic SCI, and this function was partially mediated by E-selectin.(3) During spinal cord development, the spatio-temporal expression ofβ-1, 4-GalT-V was abundant in neurons of gray matter at 3-day newborn rat (P3d), suggestingβ-1,4-GalT-V might participate in neuroal development of rat spinal cord. The expression ofβ-1,4-GalT-V after SCI was likely involved in the secondary response and neuropathic pain.(4)β-1, 4-GalT-I, V might be involved in the pain process or modulation of pain induced by CFA hindpaw injection. |
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