Expression Pattern And Effects Of Bone Morphogenetic Proteins(BMPs) After Contusive Spinal Cord Injury In The Rats

Functional Recovery from spinal cord injury (SCI) continues to be made efforts in neuroscience. The abnormality of both motor and sensory function, eg. neuropathic pain is not limited to the initial mechanical insult, but is exacerbated by the secondary mechanisms. Recent reports indicated that gliosis or glia scar formation caused by activation and proliferation of astrocytes at local lesion after SCI probably inhibited the axonal regeneration, remyelination and reestablishment of synapse. It was supposed that gliosis or glia scar formation contributed significantly to the function abnormality. However, the underlying mechanisms of gliosis or glia scar remains unclear.Some cell fate determinants genes, for example Numb,Notch-1,Shh and BMPs, express thriftily at the developing embryonic stage, Majority of them decrease gradually or even disappear with maturation. However, in the adult animals, some disappearing genes would reappear and some others express in different patterns after the central nervous system(CNS) injury.Bone morphogenetic proteins (BMPs) are such multifunctional proteins, belonging to the transforming growth factorβ(TGFβ) superfamily. They play very important roles not only in the development and morphogenesis of bone and cartilage, but also in such cell biological activities as proliferation, death and differentiation in other tissues. Recently BMPs have been regarded as crucial regulators in the development and differentiation of the nervous system, including brain, spinal cord and peripheral nerves, because they control many key processes such as the specialization of the neural ectoderm, neural crest formation, spinal cord patterning and so on. It is also believed that BMP2, BMP4 and BMP7 play crucial effects on the development of astrocytes by mediating neural stem cell differentiation into astrocytes in the embryonic proliferation. So far most experiments emphasis on the effect of BMP on the embryonic development, only a very few experiments on the adult. However the astrocytes hyperplasia in the adults after SCI would result in Gliosis or glia scar formation, which exacerbate the abnormality of nervous system. On the basis of its key roles on the development of CNS, it is hypothesized that BMPs my be one of the important cellular signals to regulate the gliosis and astroglia scar formation by facilitating the proliferation of astroglias in reponse to SCI.To elucidate the control mechanism of recovery from SCI, we performed SCI described by Scheff SW on adult rats and emphasized on the effect of BMPs on the injured spinal cord. The expression of BMPs, BMPs' receptors and the downstream signal molecule(phosphorylated Smad protein, pSmad1/5/8) in the spinal cord were detected by immunohistochemistry and west blot in the normal adult rats as well as SCI rat models at the time point of day1,3,7,14,28 and 56 after spinal cord injury. Moreover, the function of BMPs on gliosis and scar was also investigated by locally microinjection of antagonist Noggin to the lesion to block the BMPs signal. Results1. Distribution of BMPs and its receptors in the normal adult spinal cord.There were no expression of BMP2,4 in the normal adult spinal cord. All APC+ oligodendrocytes in the adult spinal cord expressed BMP7 and pSmad1/5/8. Strong pSmad1/5/8 expression was founds in the neurons in the gray matter, more in the motor neurons in the ventral horn than in the sensory neurons in the dorsal horn.BMPRⅠa was localized in a variety of cells in the adult spinal cord. The strongest expression of BMPRⅠa was observed in the ependymal cells around the central canal. Double-immunohistochemical staining showed that these BMPRⅠa immunoreactive(IR) epnedymal cells expressed 3CB2, a radial glial marker. BMPRⅠa was expressed moderately by APC+ oligodendrocytes in the gray matter and white matter, strongly in the NeuN+ motor neurons in the ventral horn, less strongly in OX-42+ microglias and part of GFAP+ astrocytes in white matter, no expression in gray matter. Double-immunostaining also showed most APC+ oliodendrocytes strongly expressed BMPRⅠb, mainly localized to the cell bodies with few short processes. Most GFAP+ astrocytes, which were radiating pendicularly from the pial surface to central canal, also moderately expressed BMPRⅠb. Some OX-42 microglias also expressed BMPRⅠb. The epedymal cells around the central canal and motor neuron in the gray matter, which strongly expressed BMPRⅠa, were not labeled by BMPRⅠb antibody. BMPRⅡexpression was found in the motor neurons and oligodendrocytes.2. Expression of BMPs and its receptors after contusive spinal cord injury in adult ratsIncreasing Expression of BMP2,4 after spinal cord injury. Both BMP2 and BMP4 increased at 3 days after spinal cord inyury, the strongest exprssion was detected in the cell bodies of hypertrophic reactive astrocytes in the gray matter and the remaining white matter, the strong expression lasted until 2 month after SCI and extended to 0.5ram rostral or cadual to the epicenter, but decreasing gradually away from the epicenter.Decreasing expression of BMP7 in oligodendrocytes after SCI. All APC+ OLGs in the normal adult expressed BMP7. At 1 day after 150 kdyne IH contusion-injury, a few OLGs in the caudal spinal cord during injury started to decrease their expression of BMP7 while most of them maintained their BMP7 expression. Starting from 3 day to 28 day post-injury (PI), some APC+ OLGs didn't express BMP7. BMP7 expression was found in astrocytes at 1 day post-injury, lasted strongly in the remaining white matter in the central lesion till 14～28 days post-injury.Decreasing expression of pSmad1/5/8 in oligodendrocytes after SCI. In the normal adult spinal cord, all APC+ OLGs expressed pSmad in their nuclei. At 1D PI, majority of pSmad positive APC+ OLGs and some pSmad negtive OLGs were found. Some APC+ OLGs remained negative expression of pSmad at 7, 14 and 30 days PI, which showed small condensed, dark-blue-labeled nuclei by Hoechst staining, suggesting apoptosis.Increasing expression of BMPs receptors. Strong expression of Ia were detected in astrocytes in the white matter of the injured spinal cord center at the day7, 14, 28, 56 PI. Double immunostain showed strongly BMPR expression in 3CB2 and GFAP double positive radial glias, and no BMPR expression in 3CB2 negative while GFAP positive glia cells.Conclusions1. Dramatically increasing expression of both BMP2 and BMP4 in the hypertrophic reactive astrocytes, decreasing expression of BMP7 and pSmad1/5/8 in oligodendrocytes in addition to the increasing expression of BMP receptor Ia in radial glia cells contribute to gliosis and scar formation, which may play a crucial effect on inhibiting the regeneration of neural axonafter spinal cord injury.2. Gliosis and hyperplasia of astrocytes are inhibited obviously by local injection of Noggin to the injured spinal cord to block the BMP signal. therefore3. BMPs signal plays a key role in recovery from SCI.The direction in the futureFurther efforts will be made on inhibiting the scar formation and promoting growth of neural axon as well as behaviors recovery using antagonist of BMP, Noggin. Hopefully it may provide the promising strategy for the treatment.