| 1. The expression and significance of β-1,4-GalT I, V ingastrocnemius muscles after sciatic nerve injuryObjective: To observe the expression of β-1,4-Galactosyltransferase I and V in ratgastrocnemius muscles after sciatic nerve crush and transection, as well as toexplore their potential biological roles during the atrophy and regeneration ofgastrocnemius muscles.Methods: Sprague-Dawley (SD) rat sciatic nerve crush and transection injury modelswere established. Sciatic nerve funtion index was detected. Gene expression of β-1,4-Galactosyltransferase I and V were detected in gastrocnemius muscles aftersciatic nerve crush and transection by Real-time fluorescence quantitative PCR aswell as in situ hybridization.Results:(1) Sciatic nerve function index assay showed that the higher sciatic nerve functionindex, the longer time, and recovered to the normal level at4weeks after sciaticnerve crush. The sciatic nerve function index keeps with-100after sciatic nervetransection.(2) In the sciatic nerve crush model, real time PCR analysis revealed that β1,4-GalT Iand V mRNAs expressed at a high level in normal gastrocnemius muscles anddecreased gradually from6hours, reached the lowest level at2weeks, thenrestored gradually to relatively normal level at4weeks after sciatic nerve crush.In situ hybridization indicated that β-1,4-GalT I and V mRNAs localized innumerous myocytes and muscle satellite cells under normal conditions and at4weeks after sciatic nerve crush.(3) In the sciatic nerve transection model, β-1,4-GalT I and V mRNAs decreasedgradually from6hours, and remained on a low level at4weeks in gastrocnemiusmuscles after sciatic nerve transection. In situ hybridization indicated that β- 1,4-GalT I and V mRNAs localized in numerous myocytes and muscle satellitecells under normal conditions and in a few muscle satellite cells at4weeks aftersciatic nerve transection.Conclusions: The expression of β-1,4-GalT I and V mRNAs are different in ratgastrocnemius muscles in different time after sciatic nerve crush and transection,which indicate that the expression of β-1,4-GalT I and V mRNAs are associatedwith the type and time of sciatic nerve injury. They localize mainly in musclesatellite cells. The expression of β-1,4-GalT I and V mRNAs are involved in theprocess of denervation and reinnervation, which suggests that β-1,4-GalT I andV mRNAs may play an important role in the muscle regeneration. 2. The expression and significance of Galβ-1,4-GlcNAcgroup in gastrocnemius muscles after sciatic nerve injuryObjective: To observe the expression and significance of Galβ-1,4-GlcNAc group inrat gastrocnemius muscles after sciatic nerve crush and transection, as well as toexplore its potential biological role during the atrophy and regeneration ofgastrocnemius muscles.Methods: Sprague-Dawley (SD) rat sciatic nerve crush and transection injury modelswere established. Lectin blot and Lectin-fluorescent staining with RCA-I, wereperformed to investigate the expression and significance of Galβ-1,4-GlcNAcgroup in gastrocnemius muscles after sciatic nerve injury.Results:(1) Lectin blot analysis showed that the expression level of the Galβ-1,4-GlcNAcgroup decreased from6hours, reached the lowest level at2weeks, and restoredto relatively normal level at4weeks after sciatic nerve crush, which wasconsistent with the expression of β1,4-GalT I and V mRNAs.(2) RCA-I lectin histochemistry analysis demonstrated that Galβ1–4GlcNAc grouplocalized in numerous membranes of myocytes and muscle satellite cells innormal and at4weeks after sciatic nerve crush, and in a few muscle satellitecells at2and4weeks after sciatic nerve transection.Conclusions: The expression level of the Galβ-1,4-GlcNAc group is different in ratgastrocnemius muscles in different time after sciatic nerve crush andtransection, which indicates that the expression of Galβ-1,4-GlcNAc group isassociated with the type and time of sciatic nerve injury. Galβ-1,4-GlcNAcgroup localizes in muscle satellite cells. The expression of Galβ-1,4-GlcNAcgroup is involved in the process of denervation and reinnervation, whichsuggests that Galβ-1,4-GlcNAc group may play an important role in the muscleregeneration. 3. The expression and significance of Kv4.2and PSD-95insciatic nerve and spinal cord during CCIObjective: To investigate the expression of the voltage gated potassium channel4.2（Kv4.2）and post-synapic density protein (PSD-95) in sciatic nerve and spinalcord during the process of chronic constriction injury(CCI) of the rat sciaticnerve. Then, observe the colocalization of Kv4.2and PSD-95in spinal cordduring this process.Methods: Sprague-Dawley (SD) rat chronic constriction injury(CCI) models wereestablished. Reverse transcription PCR and Western Blotting were used toinvestigate the changes of Kv4.2and PSD-95at mRNA and the protein levelsrespectively. The colocalization of Kv4.2with PSD-95was investigated byimmunofluorescence double staining.Results:(1) The expression of Kv4.2mRNA in sciatic nerve was moderate, decreased from1d to5d after injury and peaked at7d; And protein was not detected.(2) The expression of Kv4.2mRNA in spinal cord was high, decreased from1d to5d after injury and increased at7d, but the overall level was low; The proteinlevel was moderate in spinal cords, decreased to the lowest point at3d afterinjury, increased slowly at5d, increased significantly at14d.(3) The expression of PSD-95mRNA in sciatic nerve was moderate, increased from5d to10d, decreased at14d, but was still higher than normal; The protein leveldecreased from1d to3d after injury, increased significantly at5d, up to14d.(4) The expression of PSD-95mRNA in spinal cord was high, decreased after injury,increased from7d, reached a high point at10d; The protein level decreasedafter injury, increased significantly at10d, which was similar to Kv4.2.(5) In the lumbar spinal cord slices, immunofluorescence double staining resultsshowed that PSD-95colocalized with Kv4.2.They both colocalized with neuronin spinal dorsal horn, but not with astrocytes, which were involved in paintransmission.Conclusion: The expressions of Kv4.2and PSD-95mRNAs are different in ratsciatic nerve and spinal cord in different time after chronic constriction injury, which indicates that the expressions of Kv4.2and PSD-95mRNAs areassociated with the time of chronic constriction injury and expression sites.Kv4.2and PSD-95both colocalized with neuron in spinal dorsal horn, but notwith astrocytes, which were involved in pain transmission. All these suggest thatthe expressions of Kv4.2and PSD-95in the sciatic nerve itself and its upstreamcentral nervous system spinal cord change significantly; they may participate inthe process of chronic pain and relate to the neuronal excitability. |
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